U.S. patent application number 17/625719 was filed with the patent office on 2022-08-25 for antibodies which bind to cancer cells and target radionuclides to said cells.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Felix BORMANN, Sebastian FENN, Sofia FROST, Guy GEORGES, Alexander HAAS, Sabine IMHOF-JUNG, Christian KLEIN, Florian LIPSMEIER, Daniela MATSCHEKO, Joerg MOELLEKEN, Pablo UMANA, Barbara WEISER.
Application Number | 20220267463 17/625719 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220267463 |
Kind Code |
A1 |
UMANA; Pablo ; et
al. |
August 25, 2022 |
ANTIBODIES WHICH BIND TO CANCER CELLS AND TARGET RADIONUCLIDES TO
SAID CELLS
Abstract
The present invention relates to antibodies which bind to
antigens on target cells and which target radionuclides to said
cells, and to methods of using the same.
Inventors: |
UMANA; Pablo; (Schlieren,
CH) ; IMHOF-JUNG; Sabine; (Penzberg, DE) ;
HAAS; Alexander; (Penzberg, DE) ; KLEIN;
Christian; (Schlieren, CH) ; FROST; Sofia;
(Schlieren, CH) ; BORMANN; Felix; (Penzberg,
DE) ; GEORGES; Guy; (Penzberg, DE) ; FENN;
Sebastian; (Penzberg, DE) ; LIPSMEIER; Florian;
(Penzberg, DE) ; MATSCHEKO; Daniela; (Penzberg,
DE) ; MOELLEKEN; Joerg; (Penzberg, DE) ;
WEISER; Barbara; (Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Appl. No.: |
17/625719 |
Filed: |
July 10, 2020 |
PCT Filed: |
July 10, 2020 |
PCT NO: |
PCT/EP2020/069561 |
371 Date: |
January 7, 2022 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/44 20060101 C07K016/44; A61K 51/04 20060101
A61K051/04; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2019 |
EP |
19186135.0 |
Claims
1. A set of antibodies comprising: i) a first antibody that binds
to an antigen expressed on the surface of a target cell, and which
further comprises a VH domain of an antigen binding site for a
radiolabelled compound, but which does not comprise a VL domain of
an antigen binding site for the radiolabelled compound; and ii) a
second antibody that binds to said antigen expressed on the surface
of the target cell, and which further comprises a VL domain of an
antigen binding site for the radiolabelled compound, but which does
not comprise a VH domain of the antigen binding site for the
radiolabelled compound, wherein said VH domain of the first
antibody and said VL domain of the second antibody are together
capable of forming a functional antigen binding site for the
radiolabelled compound.
2. The set of antibodies of claim 1, wherein the first and second
antibody each comprise i) an antibody fragment comprising an
antigen binding site specific for said antigen expressed on the
surface of a target cell and ii) either a VL domain or a VH domain
of the antigen binding site for the radiolabelled compound.
3. The set of antibodies of claim 2, wherein the antibody fragment
is selected from at least one Fv, scFv, or Fab or cross-Fab
fragment.
4. The set of antibodies of claim 3, wherein the first antibody
comprises or consists of: a) a Fab fragment binding said antigen,
and b) a polypeptide comprising or consisting of i) an antibody
heavy chain variable domain (VH) of an antigen binding site for a
radiolabelled compound, or ii) an antibody heavy chain variable
domain (VH) of an antigen binding site for a radiolabelled compound
and an antibody heavy chain constant domain, wherein the C-terminus
of VH domain is fused to the N-terminus of the constant domain;
wherein the polypeptide is fused by the N-terminus of the VH domain
to the C terminus of the CL or CH1 domain of the Fab fragment; and
wherein the second antibody comprises or consists of: c) a Fab
fragment binding said antigen, and d) a polypeptide comprising or
consisting of iii) an antibody light chain variable domain (VL) of
an antigen binding site for a radiolabelled compound, or iv) an
antibody light chain variable domain (VL) of an antigen binding
site for a radiolabelled compound and an antibody light chain
constant domain, wherein the C-terminus of the VL domain is fused
to the N-terminus of the constant domain; wherein the polypeptide
is fused by the N-terminus of the VL domain to the C-terminus of
the CL or CH1 domain of the Fab fragment; and wherein antibody
heavy chain variable domain (VH) of the polypeptide of (b) and
antibody light chain variable domain (VL) of polypeptide of (d) are
together capable of forming a functional antigen binding site for
the radiolabelled compound.
5. The set of antibodies according to claim 4, wherein the
polypeptide of (b) is fused by the N-terminus of the VH domain to
the C terminus of the CL or CH1 domain of the Fab fragment of (a)
via a peptide linker; and wherein the polypeptide of (d) is fused
by the N-terminus of the VL domain to the C-terminus of the CL or
CH1 domain of the Fab fragment of (c) via a peptide linker.
6. The set of antibodies according to claim 3, wherein the first
antibody comprises a) a tandem Fab comprising two Fab fragments,
wherein the first and the second Fab fragment each bind the same
epitope of said antigen and wherein the first and the second Fab
fragment are connected via a peptide tether, wherein the first Fab
is connected via its C-terminus to the N-terminus of the second
Fab; and b) a polypeptide comprising or consisting of i) an
antibody heavy chain variable domain (VH); or ii) an antibody heavy
chain variable domain (VH) and an antibody constant domain (CH1),
wherein the C-terminus of VH domain is fused to the N-terminus of
the CH1 domain; wherein said polypeptide is fused by the N-terminus
of the VH domain to the C-terminus of the CL or CH1 domain of the
second Fab fragment; and the second antibody comprises c) a tandem
Fab comprising two Fab fragments, wherein the first and the second
Fab fragment each bind the same epitope of said antigen and wherein
the first and the second Fab fragment are connected via a peptide
tether, wherein the first Fab is connected via its C-terminus to
the N-terminus of the second Fab; and d) a polypeptide comprising
or consisting of i) an antibody light chain variable domain (VL);
or ii) an antibody light chain variable domain (VL) and an antibody
light chain constant domain (CL), wherein the C-terminus of VH
domain is fused to the N-terminus of the constant domain; wherein
said polypeptide is fused by the N-terminus of the VL domain to the
C-terminus of the CL or CH1 domain of the second Fab fragment; and
wherein antibody heavy chain variable domain (VH) of the
polypeptide of (b) and antibody light chain variable domain (VL) of
polypeptide of (d) are together capable of forming a functional
antigen binding site for the radiolabelled compound.
7. The set of antibodies according to claim 6, wherein the
polypeptide of (b) is fused by the N-terminus of the VH domain to
the C-terminus of the CL or CH1 domain of the second Fab fragment
of (a) via a peptide linker; and wherein the polypeptide of (d) is
fused by the N-terminus of the VL domain to the C-terminus of the
CL or CH1 domain of the second Fab fragment of (c) via a peptide
linker.
8. The set of antibodies according to claim 3, wherein the first
antibody comprises a) a tandem Fab comprising a first fragment and
a second fragment, wherein the first fragment is connected by its
C-terminus via a peptide tether to the N-terminus of the second
fragment, wherein the first fragment binds a first epitope of said
antigen and the second fragment binds a second epitope of said
antigen, and wherein one of the fragments selected from the first
and second fragments is a Fab and the other is a cross-Fab, b) a
polypeptide comprising or consisting of i) an antibody heavy chain
variable domain (VH); or ii) an antibody heavy chain variable
domain (VH) and an antibody heavy chain constant domain (CH1),
wherein the C-terminus of VH domain is fused to the N-terminus of
the CH1 domain; wherein said polypeptide is fused by the N-terminus
of the VH domain to the C-terminus of one of the chains of the
second fragment; and wherein the second antibody comprises c) a
tandem Fab comprising a first fragment and a second fragment,
wherein the first fragment is connected by its C-terminus to the
N-terminus of the second fragment, wherein the first fragment binds
the a first epitope of said antigen and the second fragment binds a
second epitope of said antigen, and wherein one of the fragments
selected from the first and second fragments is a Fab and the other
is a cross-Fab; and d) a polypeptide comprising or consisting of i)
an antibody light chain variable domain (VL); or ii) an antibody
light chain variable domain (VL) and an antibody light chain
constant domain (CL), wherein the C-terminus of VL domain is fused
to the N-terminus of the light chain constant domain wherein said
polypeptide is fused by the N-terminus of the VL domain to the
C-terminus of one of the chains of the second fragment; and wherein
antibody heavy chain variable domain (VH) of the polypeptide of (b)
and antibody light chain variable domain (VL) of polypeptide of (d)
are together capable of forming a functional antigen binding site
for the radiolabelled compound.
9. The set of antibodies according to claim 8, wherein the
polypeptide of (b) is fused by the N-terminus of the VL domain to
the C-terminus of one of the chains of the second fragment of (a)
via a peptide linker; and wherein the polypeptide of (d) is fused
by the N-terminus of the VL domain to the C-terminus of one of the
chains of the second fragment of (c) via a peptide linker.
10. The set of antibodies according to any one of claims 1-3,
wherein the first and second antibody each further comprise an Fc
domain.
11. The set of antibodies of claim 10, wherein the first and second
antibody each comprise i) an antibody fragment comprising an
antigen binding site specific for said antigen expressed on the
surface of a target cell, ii) and Fc region and iii) either a VL
domain or a VH domain of the antigen binding site for the
radiolabelled compound fused to the Fc region.
12. The set of antibodies of claim 10 or 11, wherein the Fc domain
is modified to reduce or eliminate effector function.
13. The set of antibodies according to any one of claims 10 to 12,
wherein the first antibody comprises or consists of: i) a complete
light chain fragment; ii) a complete heavy chain; iii) an
additional Fc chain lacking Fd; and iv) a polypeptide comprising or
consisting of the VH domain of the antigen binding site for the
radiolabeled compound; wherein the light chain of (i) and the heavy
chain of (ii) together provide an antigen binding site for said
antigen; and wherein the polypeptide comprising or consisting of
the VH domain of the antigen binding site for the radiolabeled
compound is fused by its N-terminus to the C-terminus of either
(ii) or (iii); and wherein the second antibody comprises or
consists of v) a complete light chain fragment; vi) a complete
heavy chain; vii) an additional Fc chain lacking Fd; and viii) a
polypeptide comprising or consisting of the VL domain of the
antigen binding site for the radiolabeled compound; wherein the
light chain of (v) and the heavy chain of (vi) together provide an
antigen binding site for said antigen; and wherein the polypeptide
comprising or consisting of the VL domain of the antigen binding
site for the radiolabeled compound is fused by its N-terminus to
the C-terminus of either (vi) or (vii); wherein the antibody heavy
chain variable domain (VH) of the polypeptide of (iv) and the
antibody light chain variable domain (VL) of the polypeptide of
(viii) are together capable of forming a functional antigen binding
site for the radiolabelled compound.
14. The set of antibodies according to claim 13, wherein the
polypeptide of (iv) is fused by its N-terminus to the C-terminus of
either (ii) or (iii) via a linker; and the polypeptide of (viii) is
fused by its N-terminus to the C-terminus of either (vi) or (vii)
via a linker.
15. The set of antibodies according to any one of claims 10-12,
wherein each of the first and second antibody comprise a) an Fc
domain b) at least one antibody fragment, such as an scFv, Fv, Fab
or cross-Fab fragment, comprising an antigen binding site for the
target antigen and c) a polypeptide comprising either a VL domain
or a VH domain of the antigen binding site for the radiolabelled
compound, wherein the C-terminus of the antibody fragment of (b) is
fused to the N-terminus of one chain of the Fc domain, and the
C-terminus of the polypeptide of (c) is fused to the N-terminus of
the other chain of the Fc domain.
16. The set of antibodies of claim 15, wherein the first antibody
comprises: i) a complete light chain; ii) a complete heavy chain;
iii) an additional Fc chain; and iv) a polypeptide comprising or
consisting of the VH domain of the antigen binding site for the
radiolabeled compound; wherein the light chain of (i) and the heavy
chain of (ii) together provide an antigen binding site for the
target antigen; and wherein the polypeptide comprising or
consisting of the VH domain of the antigen binding site for the
radiolabeled compound is fused by its C-terminus via a linker to
the N-terminus of (iii); and the second antibody comprises: v) a
complete light chain; vi) a complete heavy chain; vii) an
additional Fc chain; and viii) a polypeptide comprising or
consisting of the VL domain of the antigen binding site for the
radiolabeled compound; wherein the light chain of (v) and the heavy
chain of (vi) together provide an antigen binding site for the
target antigen; and wherein the polypeptide comprising or
consisting of the VL domain of the antigen binding site for the
radiolabeled compound is fused by its C-terminus via a linker to
the N-terminus of (vii).
17. The set of antibodies according to claim 16, wherein: i) the
first antibody comprises a first heavy chain of SEQ ID NO:112, a
second heavy chain of SEQ ID NO: 114 and a light chain of SEQ ID
NO: 115; and ii) the second antibody comprises a first heavy chain
of SEQ ID NO:112, a second heavy chain of SEQ ID NO: 113 and a
light chain of SEQ ID NO: 115.
18. The set of antibodies of any of claims 10 to 12, wherein the
first antibody comprises a) a first full length antibody and
consisting of two antibody heavy chains and two antibody light
chains, wherein at least one arm of the full length antibody binds
said antigen; and b) a polypeptide consisting of i) an antibody
heavy chain variable domain (VH); or ii) an antibody heavy chain
variable domain (VH) and an antibody constant domain (CH1), wherein
said polypeptide is fused by the N-terminus of the VH domain to the
C-terminus of one of the two heavy chains of said first full-length
antibody and wherein the second antibody comprises: c) a second
full length antibody and consisting of two antibody heavy chains
and two antibody light chains, wherein at least one arm of the
antibody binds to said antigen; and d) a polypeptide consisting of
i) an antibody light chain variable domain (VL); or ii) an antibody
light chain variable domain (VL) and an antibody light chain
constant domain (CL), wherein said polypeptide is fused by the
N-terminus of the VL domain to the C-terminus of one of the two
heavy chains of said second full-length antibody, wherein the
polypeptide of (b) and the polypeptide of (d) are together capable
of forming a functional antigen-binding site for the radiolabelled
compound.
19. The set of antibodies according to claim 18, wherein the
polypeptide (b) is fused by the N-terminus of the VH domain to the
C-terminus of one of the two heavy chains of said first full-length
antibody via a peptide linker; and the polypeptide of (d) is fused
by the N-terminus of the VL domain to the C-terminus of one of the
two heavy chains of said second full-length antibody via a peptide
linker.
20. The set of antibodies of claim 18 or 19, wherein each of the
first and second antibodies is bivalent for said antigen.
21. The set of antibodies of claim 20, wherein both arms of the
full length antibody bind to the same epitope of said antigen.
22. The set of antibodies of claim 18 or 19, wherein each of the
first and second antibodies is biparatopic for said antigen.
23. The set of antibodies of claim 22, wherein the two arms of the
first antibody each bind to an epitope on said antigen and bind to
different epitopes from each other; and wherein the two arms of the
second antibody each bind to an epitope on said antigen and bind to
different epitopes from each other.
24. The set of antibodies of any one of the preceding claims,
wherein the radiolabelled compound comprises radiolabelled DOTA or
a salt or functional variant thereof.
25. The set of antibodies of any one of the preceding claims,
wherein the radiolabelled compound is DOTA or a salt or functional
variant thereof radiolabelled with a Lu or Y radioisotope.
26. The set of antibodies according to claim 24 or 25, wherein the
VH domain of the antigen binding site for the radiolabelled
compound comprises (a) CDR-H1 comprising the amino acid sequence of
35; (b) CDR-H2 comprising the amino acid sequence of 36; (c) CDR-H3
comprising the amino acid sequence of 37.
27. The set of antibodies according to any one of claims 24 to 26,
wherein the VH domain of the antigen binding site for the
radiolabelled compound comprises the amino acid sequence of SEQ ID
NO: 41, or a variant thereof comprising an amino acid sequence
having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity
to SEQ ID NO: 41.
28. The set of antibodies of claim 27, wherein one or more alanine
residues are added to the C-terminus of the VH domain of the
antigen binding site for the radiolabelled compound, or wherein one
or more residues from the N-terminus of the CH1 domain are added to
the C-terminus of the VH domain of the antigen binding site for the
radiolabelled compound.
29. The set of antibodies of claim 28, wherein residues AST are
added to the C-terminus of the VH domain of the antigen binding
site for the radiolabelled compound.
30. The set of antibodies of any one of claims 24 to 29, wherein
the VL domain of the antigen binding site for the radiolabelled
compound comprises (d) CDR-L1 comprising the amino acid sequence of
38; (e) CDR-L2 comprising the amino acid sequence of 39; and (f)
CDR-L3 comprising the amino acid sequence of 40.
31. The set of antibodies of any one of claims 24 to 30, wherein
the VL domain of the antigen binding site for the radiolabelled
compound comprises an amino acid sequence of SEQ ID NO: 42 or a
variant thereof comprising an amino acid sequence having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:
42.
32. The set of antibodies of any one of claims 1 to 23, wherein the
radiolabelled compound comprises Pb-DOTAM.
33. The set of antibodies according to claim 32, wherein the
functional binding site for Pb-DOTAM binds with a Kd value of the
binding affinity of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or
less, e.g, 0.9 pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM
or less or 0.5 pM or less.
34. The set of antibodies according to claim 32 or claim 33,
wherein the functional binding site for Pb-DOTAM binds to Pb-DOTAM
and to Bi-DOTAM.
35. The set of antibodies of any one of claims 32 to 34, wherein
the VH domain of the antigen binding site for the radiolabelled
compound comprises: a) heavy chain CDR2 comprising the amino acid
sequence FIGSRGDTYYASWAKG (SEQ ID NO:2), or a variant thereof
having up to 1, 2, or 3 substitutions in SEQ ID NO: 2, wherein
these substitutions do not include Phe50, Asp56 and/or Tyr58, and
optionally also do not include Gly52 and/or Arg54; b) heavy chain
CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID
NO:3), or a variant thereof having up to 1, 2, or 3 substitutions
in SEQ ID NO: 3, wherein these substitutions do not include Glu95,
Arg96, Asp97, Pro98, and optionally also do not include Ala100C,
Tyr100D, and/or Pro100E and/or optionally also do not include
Tyr99; and a heavy chain CDR1 which is optionally: c) a heavy chain
CDR1 comprising the amino acid sequence GFSLSTYSMS (SEQ ID NO:1) or
a variant thereof having up to 1, 2, or 3 substitutions in SEQ ID
NO: 1.
36. The set of antibodies of claim 35, wherein the VH domain of the
antigen binding site for the radiolabelled compound comprises (a)
CDR-H1 comprising the amino acid sequence of GFSLSTYSMS (SEQ ID
NO:1); (b) CDR-H2 comprising the amino acid sequence of
FIGSRGDTYYASWAKG (SEQ ID NO:2) and (c) CDR-H3 comprising the amino
acid sequence of ERDPYGGGAYPPHL (SEQ ID NO:3).
37. The set of antibodies of any one of claims 32 to 36, wherein
the VH domain of the antigen binding site for the radiolabelled
compound comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 7 and SEQ ID NO 9, or a variant thereof
comprising an amino acid sequence having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 7 or SEQ ID NO:
9.
38. The set of antibodies of claim 37, wherein one or more alanine
residues are added to the C-terminus of the VH domain of the
antigen binding site for the radiolabelled compound, or wherein one
or more residues from the N-terminus of the CH1 domain are added to
the C-terminus of the VH domain of the antigen binding site for the
radiolabelled compound.
39. The set of antibodies of claim 38, wherein residues AST are
added to the C-terminus of the VH domain of the antigen binding
site for the radiolabelled compound.
40. The set of antibodies of any one of claims 32 to 39, wherein
the VL domain of the antigen binding site for the radiolabelled
compound comprises d) light chain CDR1 comprising the amino acid
sequence QSSHSVYSDNDLA (SEQ ID NO:4) or a variant thereof having up
to 1, 2, or 3 substitutions in SEQ ID NO: 4, wherein these
substitutions do not include Tyr28 and Asp32; e) light chain CDR3
comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO:6) or a
variant thereof having up to 1, 2, or 3 substitutions in SEQ ID NO:
6, wherein these substitutions do not include Gly91, Tyr92, Asp93,
Thr95c and Tyr96, and a light chain CDR2 which is optionally f)
light chain CDR 2 comprising the amino acid sequence QASKLAS (SEQ
ID NO: 5) or a variant thereof having at least 1, 2 or 3
substitutions in SEQ ID NO: 5, optionally not including Gln50.
41. The set of antibodies of claim 40, wherein the VL domain of the
antigen binding site for the radiolabelled compound comprises (d)
CDR-L1 comprising the amino acid sequence of QSSHSVYSDNDLA (SEQ ID
NO:4); (e) CDR-L2 comprising the amino acid sequence of QASKLAS
(SEQ ID NO:5); and (f) CDR-L3 comprising the amino acid sequence of
LGGYDDESDTYG (SEQ ID NO:6).
42. The set of antibodies of any one of claims 32 to 41, wherein
the VL domain of the antigen binding site for the radiolabelled
compound comprises an amino acid sequence of SEQ ID NO: 8, or a
variant thereof comprising an amino acid sequence having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:
8.
43. The set of antibodies of any one of the preceding claims,
wherein the first and second antibody bind to the same epitope of
the antigen expressed on the surface of a target cell.
44. The set of antibodies of any one of claims 1 to 42, wherein the
first antibody binds to a different epitope of the antigen from the
second antibody.
45. The set of antibodies of any one of the preceding claims,
wherein the antigen expressed on the surface of a target cell is a
tumour-associated antigen.
46. The set of antibodies of any one of the preceding claims,
wherein the antigen expressed on the surface of a target cell is
selected from the group consisting of carcinoembryonic antigen
(CEA), CD20, HER2, EGP-1 (epithelial glycoprotein-1, also known as
trophoblast-2), colon-specific antigen-p (CSAp), a pancreatic mucin
MUC1, GPRC5D and FAP.
47. The set of antibodies of any one of the preceding claims,
wherein the antigen expressed on the surface of a target cell is
selected from the group consisting of CEA, GPRC5D and FAP.
48. The set of antibodies according to claim 47, wherein: i) the
first antibody comprises a first heavy chain of SEQ ID NO: 104, a
second heavy chain of SEQ ID NO:106, wherein the C-terminal alanine
of SEQ ID NO: 106 is optional and may be absent or replaced by an
alternative C-terminal extension, and a light chain of SEQ ID NO:
107; and ii) the second antibody comprises a first heavy chain of
SEQ ID NO: 104, a second heavy chain of SEQ ID NO: 105 and a light
chain of SEQ ID NO: 107.
49. The set of antibodies according to claim 47, wherein i) the
first antibody comprises a first heavy chain of SEQ ID NO: 108, a
second heavy chain of SEQ ID NO:110, wherein the C-terminal alanine
of SEQ ID NO: 110 is optional and may be absent or replaced by an
alternative C-terminal extension, and a light chain of SEQ ID NO:
111; and ii) the second antibody comprises a first heavy chain of
SEQ ID NO: 108, a second heavy chain of SEQ ID NO:109 and a light
chain of SEQ ID NO: 111.
50. The set of antibodies of any one of claims 1 to 47, wherein the
antigen expressed on the surface of a target cell is CEA
51. The set of antibodies according to claim 50, wherein the first
antibody comprises an antigen-binding site which binds to CEA,
comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:19; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:20; and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:21; and a light
chain variable region comprising (d) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:22; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23; and (f) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
52. The set of antibodies according to any one of claims 50-51,
wherein the first antibody comprises an antigen-binding site for
CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 25, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:25.
53. The set of antibodies according to any one of claims 50-52,
wherein the first antibody comprises an antigen-binding site for
CEA comprising a VL sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 26, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:26.
54. The set of antibodies according to claim 50, wherein the first
antibody comprises an antigen-binding site which binds to CEA,
comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:43; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:44; and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:45; and a light
chain variable region comprising (d) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:46; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:47; and (0 CDR-L3 comprising the amino acid
sequence of SEQ ID NO:48.
55. The set of antibodies according to claim 50 or 54, wherein the
first antibody comprises an antigen-binding site for CEA comprising
a VH sequence comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 49, or a variant thereof comprising
an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96,
97, 98, or 99% identity to SEQ ID NO:49.
56. The set of antibodies according to any one of claim 50, 54 or
55, wherein the first antibody comprises an antigen-binding site
for CEA comprising a VL sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 50, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:50.
57. The set of antibodies according to claim 50, wherein the first
antibody comprises an antigen-binding site which binds to CEA,
comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:11; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:12; and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:13; and a light
chain variable region comprising (d) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:14; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:15; and (f) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:16.
58. The set of antibodies according to claim 50 or 57, wherein the
first antibody comprises an antigen-binding site for CEA comprising
a VH sequence comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 17, or a variant thereof comprising
an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96,
97, 98, or 99% identity to SEQ ID NO:17.
59. The set of antibodies according to claim 50, 57 or 58, wherein
the first antibody comprises an antigen-binding site for CEA
comprising a VL sequence comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 18, or a variant thereof
comprising an amino acid sequence having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:18.
60. The set of antibodies according to claim 50, wherein the first
antibody comprises an antigen-binding site which binds to CEA,
comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:59; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:60; and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:61; and a light
chain variable region comprising (d) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:62; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:63; and (0 CDR-L3 comprising the amino acid
sequence of SEQ ID NO:64.
61. The set of antibodies according to claim 50 or claim 60,
wherein the first antibody comprises an antigen-binding site for
CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 65, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:65.
62. The set of antibodies according to claim 50, 60 or 61, wherein
the first antibody comprises an antigen-binding site for CEA
comprising a VL sequence comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 66, or a variant thereof
comprising an amino acid sequence having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:66.
63. The set of antibodies according to claim 50, wherein the first
antibody comprises an antigen-binding site which binds to CEA,
comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:156; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:157 or 158; (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:159; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:160, 161 or 162; (e) CDR-L2
comprising the amino acid sequence of SEQ ID NO:163, 164 or 165;
and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:166.
64. The set of antibodies according to claim 50 or 63, wherein the
first antibody comprises an antigen-binding site which binds to
CEA, comprising a heavy chain variable region (VH) comprising an
amino acid sequence selected from SEQ ID NO: 169, 170, 171, 172,
173 or 174, or a sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98,
or 99% identity thereto; and a light chain variable region (VL)
comprising an amino acid sequence selected from SEQ ID NO: 175,
176, 177, 178, 179 or 180, or a sequence with 90, 91, 92, 93, 94,
95, 96, 97, 98, or 99% identity thereto.
65. The set of antibodies according to claim 50, 63 or 64, wherein
the first antibody comprises an antigen-binding site which binds to
CEA, comprising: (a) a VH domain comprising an amino acid sequence
of SEQ ID NO:169 and a VL domain comprising an amino acid sequence
of SEQ ID NO:179, or (b) a VH domain comprising an amino acid
sequence of SEQ ID NO:173 and a VL domain comprising an amino acid
sequence of SEQ ID NO:179, or (c) a VH domain comprising an amino
acid sequence of SEQ ID NO:170 and a VL domain comprising an amino
acid sequence of SEQ ID NO:179, or (d) a VH domain comprising an
amino acid sequence of SEQ ID NO:174 and a VL domain comprising an
amino acid sequence of SEQ ID NO:178, or (e) a VH domain comprising
an amino acid sequence of SEQ ID NO:173 and a VL domain comprising
an amino acid sequence of SEQ ID NO:178, or (f) a VH domain
comprising an amino acid sequence of SEQ ID NO:171 and a VL domain
comprising an amino acid sequence of SEQ ID NO:178, or (g) a VH
domain comprising an amino acid sequence of SEQ ID NO:169 and a VL
domain comprising an amino acid sequence of SEQ ID NO:178.
66. The set of antibodies according to any one of claims 50 to 65,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising a heavy chain variable region comprising
(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; (b)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; and (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:21; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:22; (e) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:23; and (f) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:24.
67. The set of antibodies according to any one of claims 50-66,
wherein the second antibody comprises an antigen-binding site for
CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 25, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:25.
68. The set of antibodies according to any one of claims 50-67,
wherein the second antibody comprises an antigen-binding site for
CEA comprising a VL sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 26, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:26.
69. The set of antibodies according to any one of claims 50 to 65,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising a heavy chain variable region comprising
(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:43; (b)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:44; and (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:45; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:46; (e) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:47; and (f) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:48.
70. The set of antibodies according to any one of claim 50 to 65 or
69, wherein the second antibody comprises an antigen-binding site
for CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 49, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:49.
71. The set of antibodies according to any one of claim 50 to 65,
69 or 70, wherein the second antibody comprises an antigen-binding
site for CEA comprising a VL sequence comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 50, or a
variant thereof comprising an amino acid sequence having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID
NO:50.
72. The set of antibodies according to any one of claims 50 to 65,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising a heavy chain variable region comprising
(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:11; (b)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:12; and (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:13; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:14; (e) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:15; and (f) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:16.
73. The set of antibodies according to any one of claim 50 to 65 or
72, wherein the second antibody comprises an antigen-binding site
for CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 17, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:17.
74. The set of antibodies according to any one of claim 50 to 65,
72 or 73, wherein the second antibody comprises an antigen-binding
site for CEA comprising a VL sequence comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 18, or a
variant thereof comprising an amino acid sequence having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID
NO:18.
75. The set of antibodies according to any one of claims 50 to 65,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising a heavy chain variable region comprising
(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:59; (b)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:60; and (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:61; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:62; (e) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:63; and (f) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:64.
76. The set of antibodies according to any one of claim 50 to 65 or
75, wherein the second antibody comprises an antigen-binding site
for CEA comprising a VH sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 65, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:65.
77. The set of antibodies according to claim 50 to 65, 75 or 76,
wherein the second antibody comprises an antigen-binding site for
CEA comprising a VL sequence comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 66, or a variant
thereof comprising an amino acid sequence having at least 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:66.
78. The set of antibodies according to claims 50 to 65, wherein the
second antibody comprises an antigen-binding site which binds to
CEA, comprising a heavy chain variable region comprising (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:156; (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:157 or 158; (c)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:159; and a
light chain variable region comprising (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:160, 161 or 162; (e) CDR-L2
comprising the amino acid sequence of SEQ ID NO:163, 164 or 165;
and (0 CDR-L3 comprising the amino acid sequence of SEQ ID
NO:166.
79. The set of antibodies according to claim 50 to 65 or 78,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising a heavy chain variable region (VH)
comprising an amino acid sequence selected from SEQ ID NO: 169,
170, 171, 172, 173 or 174, or a sequence with 90, 91, 92, 93, 94,
95, 96, 97, 98, or 99% identity thereto; and a light chain variable
region (VL) comprising an amino acid sequence selected from SEQ ID
NO: 175, 176, 177, 178, 179 or 180, or a sequence with 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99% identity thereto.
80. The set of antibodies according to claim 50 to 65, 78 or 79,
wherein the second antibody comprises an antigen-binding site which
binds to CEA, comprising: (a) a VH domain comprising an amino acid
sequence of SEQ ID NO:169 and a VL domain comprising an amino acid
sequence of SEQ ID NO:179, or (b) a VH domain comprising an amino
acid sequence of SEQ ID NO:173 and a VL domain comprising an amino
acid sequence of SEQ ID NO:179, or (c) a VH domain comprising an
amino acid sequence of SEQ ID NO:170 and a VL domain comprising an
amino acid sequence of SEQ ID NO:179, or (d) a VH domain comprising
an amino acid sequence of SEQ ID NO:174 and a VL domain comprising
an amino acid sequence of SEQ ID NO:178, or (e) a VH domain
comprising an amino acid sequence of SEQ ID NO:173 and a VL domain
comprising an amino acid sequence of SEQ ID NO:178, or (f) a VH
domain comprising an amino acid sequence of SEQ ID NO:171 and a VL
domain comprising an amino acid sequence of SEQ ID NO:178, or (g) a
VH domain comprising an amino acid sequence of SEQ ID NO:169 and a
VL domain comprising an amino acid sequence of SEQ ID NO:178.
81. The set of antibodies according to claim 50, wherein the first
antibody is an antibody according to any one of claims 57 to 59 and
the second antibody is an antibody according to any one of claims
66 to 68.
82. The set of antibodies according claim 1, wherein the first
antibody comprises a first heavy chain of SEQ ID NO: 28, a second
heavy chain of SEQ ID NO: 32 and a light chain of SEQ ID NO:
34.
83. The set of antibodies according to claim 82, wherein one or
more alanine residues is added to the C-terminus of SEQ ID NO: 32,
or wherein the sequence AST is added to the C-terminus of SEQ ID
NO: 32.
84. The set of antibodies according to claim 1, wherein the first
antibody comprises a first heavy chain of SEQ ID NO: 51, and second
heavy chain of SEQ ID NO: 52 and a light chain of SEQ ID NO:
54.
85. The set of antibodies according to claim 84, wherein one or
more alanine residues is added to the C-terminus of SEQ ID NO:52 or
wherein the sequence AST is added to the C-terminus of SEQ ID NO:
32.
86. The set of antibodies according to claim 1, wherein the first
antibody comprises a first heavy chain of SEQ ID NO: 86, a second
heavy chain of SEQ ID NO: 87 wherein the C-terminal AST residues
are optional and may be absent or replaced by a different
C-terminal extension, and a light chain of SEQ ID NO: 89.
87. The set of antibodies according to claim 1, wherein the first
antibody comprises a first heavy chain of SEQ ID NO: 93, a second
heavy chain of SEQ ID NO: 94 wherein the C-terminal AST residues
are optional and may be absent or replaced by a different
C-terminal extension, and a light chain of SEQ ID NO: 96.
88. The set of antibodies according to claim 1, or 82-87 wherein
the second antibody comprises a first heavy chain of SEQ ID NO: 30,
a second heavy chain of SEQ ID NO: 33 and a light chain of SEQ ID
NO: 34.
89. The set of antibodies according to claim 1, or 82-87, wherein
the second antibody comprises a first heavy chain of SEQ ID NO: 55,
and second heavy chain of SEQ ID NO: 56 and a light chain of SEQ ID
NO: 58.
90. The set of antibodies according to claim 1 or 82-87, wherein
the second antibody comprises a first heavy chain of SEQ ID NO: 83,
and second heavy chain of SEQ ID NO: 84 and a light chain of SEQ ID
NO: 89.
91. The set of antibodies according to claim 1 or 82-87, wherein
the second antibody comprises a first heavy chain of SEQ ID NO:90,
and second heavy chain of SEQ ID NO: 91 and a light chain of SEQ ID
NO: 96.
92. A set of nucleic acids expressing the set of antibodies of any
one of the preceding claims.
93. An expression vector or set of expression vectors comprising
the set of nucleic acids according to claim 92.
94. A host cell or set of host cells comprising the expression
vector or set of expression vectors of claim 93.
95. A method of pretargeting radioimmunotherapy, comprising i)
administering to a subject the set of antibodies according to any
one of claims 1 to 91, wherein the first and second antibodies are
administered simultaneously or sequentially, in either order,
wherein the antibodies bind to the target antigen and localise to
the surface of a cell expressing the target antigen; and wherein
association of the first and second antibody forms a functional
binding site for the radiolabelled compound; and ii) subsequently
administering a radiolabelled compound, wherein the radiolabelled
compound binds to functional binding site for the radiolabelled
compound.
96. The method of claim 95, wherein the method does not comprise a
step of administering a clearing or blocking agent.
97. The method of claim 95 or 96, wherein the subject is human.
98. The method of any one of claims 95 to 97, wherein the target
antigen is a cancer- or tumour-associated antigen and the method is
a method of radioimmunotherapy of a tumour or cancer.
99. The set of antibodies according to any one of claims 1 to 91,
for use in a method of pretargeting radioimmunotherapy according to
any one of claims 95 to 98.
100. A method of targeting a radioisotope to a tissue or organ for
radioimaging comprising: i) administering to a subject a set of
antibodies according to any one of claims 1 to 91, wherein the
first and second antibodies are administered simultaneously or
sequentially, in either order, wherein the antibodies bind to a
target antigen and localise to the surface of a cell expressing the
target antigen, wherein association of the first and second
antibody forms a functional binding site for the radiolabelled
compound; and ii) subsequently administering a radiolabelled
compound, wherein the radiolabelled compound binds to the
functional binding site for the radiolabelled compound.
101. The method of claim 100, wherein the method does not comprise
a step of administering a clearing or blocking agent.
102. The method of claim 100 or 101, wherein the method further
comprises a step of imaging.
103. The method of claim 102, wherein the target antigen is a
cancer or tumour-associated antigen and the method is a method of
imaging tumours or cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to antibodies which bind to
antigens on target cells and which target radionuclides to said
cells, and to methods of using the same.
BACKGROUND
[0002] Monoclonal antibodies have been developed to target drugs to
cancer cells. By conjugating a toxic agent to an antibody which
binds to a tumour-associated antigen, there is the potential to
provide more specific tumour killing with less damage to
surrounding tissues.
[0003] In pre-targeted radioimmunotherapy (PRIT), use is made of an
antibody construct which has affinity for the tumour-associated
antigen on the one hand and for a radiolabelled compound on the
other. In a first step, the antibody is administered and localises
to tumour. Subsequently, the radiolabelled compound is
administered. Because the radiolabelled compound is small, it can
be delivered quickly to the tumour and is fast-clearing, which
reduces radiation exposure outside of the tumour (Goldenberg et al
Theranostics 2012, 2(5), 523-540). A similar procedure can also be
used for imaging. Pre-targeting can make use of a bispecific
antibody or systems using avidin-biotin, although the latter has
the disadvantage that avidin/streptavidin is immunogenic.
[0004] Methods of pre-targeted radioimmunotherapy or imaging
commonly make use of a clearing or blocking agent, which is
administered between the step of administering the antibody and the
step of administering the radiolabelled compound. The purpose is to
clear antibody from the blood and/or to block the binding site of
the circulating antibody for the radiolabelled compound (see for
instance Karacay et al, Bioconj. Chem., 13(5), 1054-1070 (2002)).
The use of a clearing or blocking agent allows for sufficient
levels of radioactivity to be administered for an efficient
treatment while limiting adverse toxicity, but the timing and
dosage must be chosen with care. Thus, the use of a clearing phase
is a complicating aspect in pre-targeting methods.
SUMMARY
[0005] The present invention provides sets of antibodies useful in
pre-targeting methods, and methods of using the same.
[0006] In one aspect, the present invention provides a set of
antibodies comprising: [0007] i) a first antibody that binds to an
antigen expressed on the surface of a target cell, and which
further comprises a V.sub.H domain of an antigen binding site for a
radiolabelled compound, but which does not comprise a V.sub.L
domain of an antigen binding site for the radiolabelled compound;
and [0008] ii) a second antibody that binds to the antigen
expressed on the surface of the target cell, and which further
comprises a V.sub.L domain of an antigen binding site for the
radiolabelled compound, but which does not comprise a V.sub.H
domain of the antigen binding site for the radiolabelled
compound,
[0009] wherein said V.sub.H domain of the first antibody and said
V.sub.L domain of the second antibody are together capable of
forming a functional antigen binding site for the radiolabelled
compound.
[0010] Neither the first nor the second antibody comprise, on their
own, a functional antigen binding site for a radiolabelled
compound. The first antibody has only a V.sub.H domain from the
functional binding site for the radiolabelled compound, and not the
V.sub.L domain. The second antibody has only the V.sub.L domain,
and not the V.sub.H domain.
[0011] A functional antigen binding site for the radiolabelled
compound is formed when the V.sub.H and V.sub.L domains of the
first and second antibodies are associated. This may occur, for
example, when the first and second antibodies are bound to the same
individual target cell or to adjacent cells.
[0012] The first and second antibodies described herein may be
referred to herein as "single domain split antibodies", "split
antibodies" or "demibodies". The V.sub.H and V.sub.L domain which
together form an antigen binding site capable of binding to the
radiolabelled compound are split between two antibodies, and not
present as part of the same antibody.
[0013] The split domain format means that the radiolabelled
compound cannot bind to either the first antibody on its own or to
the second antibody on its own. In the blood, there is little or no
stable association between the first and the second antibody, and
so little or no stable binding of the radiolabelled compound.
[0014] An antigen expressed on the surface of a target cell may be
referred to herein as a "target antigen" or "TA". According to the
present invention, the first and the second antibody described
above have a binding site for the same target antigen. (For the
avoidance of doubt, where it stated that the antibodies bind the
same target antigen, this means that they have a binding site
capable of binding to the same target antigen and includes the
possibility that the antibodies may bind to two individual antigen
molecules that are the same as each other). For example, in one
embodiment, both the first and the second antibody bind to CEA.
[0015] In some embodiments, the first and second antibody may bind
to (have a binding site for) the same epitope of the target
antigen. In other embodiments, the first antibody may bind to (have
a binding site for) a different epitope of the target antigen from
the second antibody.
[0016] In some embodiments, the first and second antibody may
comprise the same antigen binding site for the target antigen. That
is, they may comprise an antigen binding site capable of binding to
the target antigen, comprising a V.sub.L and V.sub.H sequence,
where the V.sub.L and V.sub.H sequences forming this antigen
binding site are the same in the first and in the second
antibodies.
[0017] In some embodiments, each of the first and the second
antibodies are bivalent for the target antigen. In some embodiments
they are each bivalent and monospecific for an epitope. In other
embodiments, each of the first and second antibodies are
biparatopic for the target antigen, i.e., the first and the second
antibodies each have binding sites for two different epitopes of
the target antigen.
[0018] In some embodiments, it may be preferred that the first
and/or second antibody comprise an Fc region. The presence of an Fc
region has benefits in the context of radioimmunotherapy and
radioimaging, e.g. prolonging the protein's circulating half-life
and/or resulting in higher tumour uptake than may be observed with
smaller fragments. The "split domain" format described herein may
be particularly advantageous in this context, as it mitigates
against the greater possibility of association with radiolabelled
compound that would otherwise occur due to the prolonged presence
of the circulating antibody.
[0019] In some embodiments, the Fc domain is modified to reduce or
eliminate effector function.
[0020] In another aspect, the present invention provides a
pharmaceutical composition comprising the set of antibodies as
described herein. In another aspect, the present invention provides
a kit comprising two separate pharmaceutical compositions, each
comprising one of the antibodies described herein (i.e., the first
and second antibody respectively).
[0021] In a further aspect, the present invention relates to a
polynucleotide or set of polynucleotides encoding any of the
antibodies or sets of antibodies described herein. In another
aspect, the present invention relates to a vector or set of vectors
comprising said polynucleotide or polynucleotides, optionally an
expression vector or set of expression vectors. In a further object
the present invention relates to a prokaryotic or eukaryotic host
cell or a set of host cells comprising a vector or set of vectors
of the present invention. In addition there is provided a method of
producing an antibody comprising culturing the host cell(s) so that
the antibody is produced.
[0022] In some embodiments, antibodies as described herein find use
in a method of pre-targeted radioimmunotherapy (PRIT) or in a
method of pre-targeted radioimaging.
[0023] In one aspect, the present invention provides a method of
pre-targeted radioimmunotherapy which comprises: [0024] i)
administering to a subject a first antibody and a second antibody
as described above; and [0025] ii) subsequently administering to
said subject a radiolabelled compound.
[0026] In another aspect, the present invention provides a first
and a second antibody described above for use in a method of
treatment comprising administering the first antibody and the
second antibody to a subject, and subsequently administering to
said subject a radiolabelled compound. In another aspect, the
invention provides a first antibody as described above for use in a
method of treatment comprising administering the first antibody and
the second antibody to a subject, and subsequently administering to
said subject a radiolabelled compound. In another aspect the
invention provides the second antibody as described above for use
in a method of treatment comprising administering the first
antibody and the second antibody to a subject, and subsequently
administering to said subject a radiolabelled compound.
[0027] In another aspect, the present invention provides a method
of radioimaging which comprises: [0028] i) administering to a
subject a first and a second antibody as described herein, wherein
the antibodies bind to the target antigen and localise to the
surface of a cell expressing the target antigen; [0029] ii)
subsequently administering a radiolabelled compound; and optionally
[0030] iii) imaging the tissue or organ where the radionuclide has
localised.
[0031] In another aspect, the present invention provides a first
and a second antibody as described herein for use in a method of
diagnosis carried out on the human or animal body, wherein the
method comprises [0032] i) administering to subject a first and a
second antibody as described herein, wherein the antibodies bind to
the target antigen and localise to the surface of a cell expressing
the target antigen; [0033] ii) subsequently administering a
radiolabelled compound; and optionally [0034] iii) imaging the
tissue or organ where the radionuclide has localised.
[0035] The imaging step may be followed by a step of forming a
diagnosis and optionally a step of delivering that diagnosis to the
subject. In some embodiments the method may further comprise
determining an appropriate treatment and optionally administering
that treatment to the subject.
[0036] In each of the above methods/uses, binding of the first and
the second antibody to the same or adjacent target cells results in
association of the V.sub.H and V.sub.L domains of an antigen
binding site for a radiolabelled compound and the formation of a
functional antigen binding site for the radiolabelled compound.
Thus, after administration of the radiolabelled compound, the
radiolabelled compound binds to the functional antigen binding site
formed by association of the V.sub.H and V.sub.L.
[0037] In any of the methods and uses described herein, the first
and second antibodies can be administered simultaneously or
sequentially, in either order.
[0038] Frequently in the art, methods of PRIT or radioimaging
involve a clearing step. The clearing step comprises administering
an agent between the administration of the antibody and the
administration of the radiolabelled compound, wherein the agent
increases the rate of removal of the antibody from blood and/or
blocks binding of radiolabelled compound to the antibody.
[0039] In an embodiment of the methods and uses described herein,
the method does not comprise a clearing step. That is, it does not
comprise a step of administering a clearing agent or a blocking
agent between the administration of the first and second antibodies
and the administration of radiolabelled compound (i.e., after the
administration of the antibodies but before administration of the
radiolabelled compound). In another embodiment, no agent is
administered between the administration of the first and second
antibodies and the administration of radiolabelled compound, other
than optionally a radiosensitizer, immunotherapeutic and/or a
chemotherapeutic agent. In another embodiment, no agent is
administered between the administration of the first and second
antibodies and the administration of radiolabelled compound.
[0040] In some embodiments, the antibodies described herein may be
administered as part of a combination therapy. For example, they
may be administered in combination with one or more
radiosensitizers, immunotherapeutics and/or chemotherapeutic
agents: the radiosensitizer, immunotherapeutic or chemotherapeutic
agent and the antibodies may be administered simultaneously or
sequentially, in either order.
[0041] The methods of radioimaging and radioimmunotherapy described
herein may optionally be combined as discussed further herein.
[0042] In a further aspect, the present invention provides a kit
comprising: [0043] i) a first and a second antibody as described
herein; [0044] ii) a radiolabelled compound which binds to the
antigen binding site formed by association of the first and the
second antibody.
[0045] Optionally the kit may exclude (i.e., does not comprise) a
clearing agent or a blocking agent as described herein.
[0046] Optionally the kit may further comprise a radiosensitizer,
immunotherapeutic or chemotherapeutic agent.
[0047] In some embodiments, the first and the second antibody may
be present in the same pharmaceutical composition. In other
embodiments, the first and second antibody may be present in
separate pharmaceutical compositions. In some embodiments, the
radiolabelled compound is present in a pharmaceutical composition
separate from the antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0048] FIG. 1 shows the schematic structure of a target antigen
(TA)-DOTAM bispecific antibody (TA-DOTAM BsAb) belonging to the
comparative examples, and exemplary TA-split-DOTAM-VH/VL antibodies
according to the invention.
[0049] FIG. 2 is a schematic diagram showing the assembly of the
split-VH/VL DOTAM binder on tumour cells. The TA-split-DOTAM-VH/VL
antibodies will not significantly bind .sup.212Pb-DOTAM unless
bound to tumour antigen (TA) on targeted cells, where the two
domains of the DOTAM binder are assembled.
[0050] FIG. 3 shows a schematic overview of an example of the
Three-Step TA-PRIT concept, involving use of a clearing agent.
[0051] FIG. 4 shows a schematic overview of an example of the
Two-Step TA-PRIT concept, in which a clearing agent is not
used.
[0052] FIG. 5 shows binding of split antibodies to MKN45 cells to
demonstrate CEA binding competence. Detection of antibodies is done
using human IgG specific secondary antibodies
[0053] FIG. 6 shows binding of split antibodies to MKN45 cells to
demonstrate DOTAM binding competence. Detection of antibodies is
done using Pb-DOTAM-FITC.
[0054] FIG. 7A shows an exemplary protocol for two-step PRIT with a
CEA-split-DOTAM-VH/VL, carried out in in SCID mice carrying SC
BxPC3 tumours (h=hours, d=days, w=weeks).
[0055] FIG. 7B shows an exemplary protocol for a three-step PRIT
control, carried out in SCID mice carrying SC BxPC3 tumours
(h=hours, d=days, w=weeks).
[0056] FIG. 8 shows the biodistribution of pretargeted
.sup.212Pb-DOTAM in SCID mice carrying SC BxPC3 tumors, 6 hours
after injection of .sup.212Pb-DOTAM, pretargeted either by
CEA-split-DOTAM-VH alone, CEA-split-DOTAM-VL alone, or the two
complimentary antibodies combined, or using standard three-step
PRIT (% ID/g .+-.SD, n=4).
[0057] FIG. 9 shows CEA-Split-DOTAM-VH/VL phaiivacokinetics after
IV injection in SCID mice.
[0058] FIG. 10 shows the experimental design of protocol 158,
comprising CEA-PRIT in 2 (top) or 3 steps (bottom) in SCID mice
carrying SC BxPC3 tumors. *CEA split DOTAM BsAb dose adjusted to
compensate for hole/hole impurities in 2/4 constructs.
[0059] FIG. 11 shows the biodistribution of pretargeted
.sup.212Pb-DOTAM in SCID mice carrying SC BxPC3 tumors (6 h p.i.).
The distribution is of .sup.212Pb in tumour-bearing SCID mice, 6
hours after injection of .sup.212Pb-DOTAM, pretargeted by CEA-DOTAM
BsAb or bi-paratopic combinations of CEA-split-DOTAM antibodies.
The radioactive content in organs and tissues is expressed as
average % ID/g .+-.SD (n=4).
[0060] FIG. 12 shows the experimental schedule of protocol 160,
comprising one cycle of 3-step CEA-PRIT (top), 2-step CEA-PRIT
(middle), or 1-step CEA-RIT in SCID mice carrying SC BxPC3 tumors.
Biodistribution (BD) scouts were euthanized 24 hours after the
radioactive injection, whereas mice in the efficacy groups were
maintained and monitored carefully until the termination criteria
were reached.
[0061] FIG. 13 shows biodistribution of pretargeted
.sup.212Pb-DOTAM and .sup.212Pb-DOTAM-CEA-DOTAM in SCID mice
carrying SC BxPC3 tumors (24 h p.i.). The distribution is of
.sup.212Pb in tumor-bearing SCID mice 24 hours after injection of
CEA-DOTAM-pretargeted .sup.212Pb-DOTAM or pre-incubated
.sup.212Pb-DOTAM-CEA-DOTAM. The radioactive content in organs and
tissues is expressed as average % ID/g .+-.SD (n=3).
[0062] FIG. 14 shows tumor growth averages with standard error for
PRIT-treated groups and control (groups A-E) in the BxPC3 model
(n=10). Curves were truncated at n<5. Dotted vertical lines
indicate .sup.212Pb-DOTAM administration (20 .mu.Ci) for some or
all groups, according to the study design.
[0063] FIG. 15 shows individual tumor growth curves for
PRIT-treated groups and control (groups A-E) in the BxPC3 model
(n=10). Dotted vertical lines indicate administration of
.sup.212Pb-labeled compounds (20 .mu.Ci).
[0064] FIG. 16 shows average body weight loss in mice treated with
CEA-PRIT and CEA-RIT (groups A-E, n=10) in the BxPC3 model. Curves
were truncated at n<5. Dotted vertical lines indicate
administration of .sup.212Pb-labeled compounds for some or all
groups, according to the study design.
[0065] FIG. 17 shows the experimental design of protocol 175,
comprising two-step CEA-PRIT in SCID mice carrying SC BxPC3 tumors,
with sacrifice and necropsy 24 hours after the .sup.212Pb-DOTAM
injection. The CEA-split-DOTAM-VH-AST dose was adjusted to
compensate for hole/hole impurities.
[0066] FIG. 18 shows distribution of .sup.212Pb in tumor-bearing
SCID mice 24 hours after injection of .sup.212Pb-DOTAM, pretargeted
by CEA-split-DOTAM-VH/VL antibodies (protocol 175). The radioactive
content in organs and tissues is expressed as average % ID/g .+-.SD
(n=4).
[0067] FIG. 19 shows the experimental design of protocol 185,
comprising two-step CEA-PRIT in SCID mice carrying SC BxPC3 tumors,
with sacrifice and necropsy 6 hours after the .sup.212Pb-DOTAM
injection. The CEA-split-DOTAM-VH-AST (CH1A1A) dose was adjusted to
compensate for hole/hole impurities.
[0068] FIG. 20 shows distribution of .sup.212Pb in tumor-bearing
SCID mice 6 hours after injection of .sup.212Pb-DOTAM, pretargeted
by CEA-split-DOTAM-VH/VL antibodies (protocol 185). The radioactive
content in organs and tissues is expressed as average % ID/g .+-.SD
(n=5).
[0069] FIG. 21 shows distribution of CEA-split-DOTAM-VH/VL pairs
(VH and VL antibodies combined) in two selected SC BxPC3 tumors 7
days after injection. A and B show sections of a tumor from mouse
A3, injected with CEA-split-DOTAM-VH/VL targeting T84.66, where A
shows the CEA expression, and B shows the corresponding
CEA-split-DOTAM-VH/VL distribution. C and D show tumor sections
from mouse C5, injected with CEA-split-DOTAM-VH/VL targeting
CH1A1A: C showing the CEA expression and D the corresponding
CEA-split-DOTAM-VH/VL distribution.
[0070] FIG. 22 shows the experimental design of protocol 189,
comprising two-step CEA-PRIT in SCID mice carrying SC BxPC3 tumors,
with sacrifice and necropsy 6 hours after the .sup.212Pb-DOTAM
injection. The CEA-split-DOTAM-VH-AST (CH1A1A) dose was adjusted to
compensate for hole/hole impurities.
[0071] FIG. 23 shows distribution of .sup.212Pb in tumor-bearing
SCID mice 6 hours after injection of .sup.212Pb-DOTAM, pretargeted
by bi-paratopic pairs of CEA-split-DOTAM-VH/VL antibodies (T84.66
and CH1A1A), compared with the positive control (CH1A1A only). The
radioactive content in organs and tissues is expressed as average %
ID/g .+-.SD.
[0072] FIG. 24 shows mean Flurescence Intensity (MFI) as determined
by FACS for SPLIT antibodies. Binding of Pb-DOTA-FITC determined by
FACS can only be shown for a co-incubation of both SPLIT antibodies
with Pb-DOTA-FITC. Single SPLIT antibodies did not give rise to a
significant signal.
[0073] FIG. 25A-C shows exemplary formats of antibodies as
described herein.
[0074] FIG. 26 shows results from example 11, experiment 1,
assessing binding of individual TA-split-DOTAM-VH and
TA-split-DOTAM-VL antibodies to biotinylated DOTAM captured on a
chip.
[0075] FIG. 27 shows results from example 11, experiment 2,
assessing binding of DOTAM to individual TA-split-DOTAM-VH and
TA-split-DOTAM-VL antibodies captured on a chip.
[0076] FIG. 28 shows results from example 11, experiment 3,
assessing binding of DOTAM to TA-split-DOTAM-VH/VL antibodies
(antibody pairs), captured on a chip.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0077] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
aspects, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some aspects, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0078] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (K.sub.D).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary methods for measuring binding affinity are described in
the following.
[0079] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more complementary determining
regions (CDRs), compared to a parent antibody which does not
possess such alterations, such alterations resulting in an
improvement in the affinity of the antibody for antigen.
[0080] The term "an antibody that binds to an antigen expressed on
the surface of a target cell" refers to an antibody that is capable
of binding said antigen with sufficient affinity such that the
antibody is useful as a diagnostic and/or therapeutic agent in
targeting said antigen. In one aspect, the extent of binding of the
antibody to an unrelated, non antigen protein is less than about
10% of the binding of the antibody to the antigen as measured,
e.g., by surface plasmon resonance (SPR). In certain aspects, an
antibody that binds to an antigen expressed on the surface of a
target cell has a dissociation constant (K.sub.D) of .ltoreq.1
.mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g., 10.sup.-8 M or less,
e.g., from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). An antibody is said to "specifically bind" to an
antigen expressed on the surface of a target cell when the antibody
has a K.sub.D of 1 .mu.M or less. In certain aspects, the antibody
binds to an epitope of said antigen that is conserved among said
antigen from different species.
[0081] The terms "an antigen binding site for a radiolabelled
compound" or "a functional antigen binding site for a radiolabelled
compound" refer to an antigen binding site comprising VH and a VL
domain, capable of binding to the radiolabelled compound with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent to associate the radiolabelled
compound with the antibody. In one aspect, the extent of binding of
the antigen binding site to an unrelated, non antigen-compound is
less than about 10% of the binding of the antibody to the
radiolabelled compound as measured, e.g., by surface plasmon
resonance (SPR). In certain aspects, an antigen binding site that
binds to a radiolabelled compound has a dissociation constant (KD)
of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM,
.ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g.,
10.sup.-8 M or less, e.g., from 10.sup.-8 M to 10.sup.-13 M, e.g.,
from 10.sup.-9 M to 10.sup.-13 M). It may be preferred that it has
a KD of 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g, 0.9
pM or less, 0.8 pM or less, 0.7 pM or less, 0.6 pM or less or 0.5
pM or less. For instance, the functional binding site may bind the
radiolabelled compound with a Kd of about 1 pM-1 nM, e.g., about
1-10 pM, 1-100 pM, 5-50 pM, 100-500 pM or 500 pM-1 nM. An antigen
binding site is said to "specifically bind" to a radiolabelled
compound when the antigen binding site has a KD of 1 .mu.M or
less.
[0082] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0083] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab,
cross-Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear
antibodies; single-chain antibody molecules (e.g., scFv, and
scFab); single domain antibodies (dAbs); and multispecific
antibodies formed from antibody fragments. For a review of certain
antibody fragments, see Holliger and Hudson, Nature Biotechnology
23:1126-1136 (2005). The term "Fab fragment" thus refers to an
antibody fragment comprising a light chain comprising a VL domain
and a CL domain, and a heavy chain fragment comprising a VH domain
and a CH1 domain. "Fab' fragments" differ from Fab fragments by the
addition of residues at the carboxy terminus of the CH1 domain
including one or more cysteines from the antibody hinge region. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046. The term "cross-Fab fragment" or "xFab
fragment" or "crossover Fab fragment" refers to a Fab fragment,
wherein either the variable regions or the constant regions of the
heavy and light chain are exchanged. A cross-Fab fragment comprises
a polypeptide chain composed of the light chain variable region
(VL) and the heavy chain constant region 1 (CH1), and a polypeptide
chain composed of the heavy chain variable region (VH) and the
light chain constant region (CL). Asymmetrical Fab arms can also be
engineered by introducing charged or non-charged amino acid
mutations into domain interfaces to direct correct Fab pairing. See
e.g., WO 2016/172485.
[0084] A "single-chain variable fragment" or "scFv" is a fusion
protein of the variable domains of the heavy (VH) and light chains
(VL) of an antibody, connected by a peptide linker. In particular,
the linker is a short polypeptide of 10 to 25 amino acids and is
usually rich in glycine for flexibility, as well as serine or
threonine for solubility, and can either connect the N-terminus of
the VH with the C-terminus of the VL, or vice versa. This protein
retains the specificity of the original antibody, despite removal
of the constant regions and the introduction of the linker. For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.
[0085] The term "blocking agent" refers to an agent which blocks
the binding of an effector molecule, in particular the
radiolabelled compound, to a functional binding site for that
effector molecule. Generally said blocking agent binds to the
functional binding site for the effector molecule, e.g.,
specifically binds to the said functional binding site.
[0086] The term "clearing agent" refers to an agent which increases
the rate of clearance of an antibody from the circulation of the
subject. Generally the clearing agent binds to the antibody, e.g.,
specifically binds to the antibody.
[0087] The term "clearing step" or "clearing phase" as used herein
encompasses the use of either a blocking agent or a clearing agent.
Some agents can function as both a clearing and as a blocking
agent.
[0088] The term "epitope" denotes the site on an antigen, either
proteinaceous or non-proteinaceous, to which an antibody binds.
Epitopes can be formed both from contiguous amino acid stretches
(linear epitope) or comprise non-contiguous amino acids
(conformational epitope), e.g., coming in spatial proximity due to
the folding of the antigen, i.e. by the tertiary folding of a
proteinaceous antigen. Linear epitopes are typically still bound by
an antibody after exposure of the proteinaceous antigen to
denaturing agents, whereas conformational epitopes are typically
destroyed upon treatment with denaturing agents. An epitope
comprises at least 3, at least 4, at least 5, at least 6, at least
7, or 8-10 amino acids in a unique spatial conformation.
[0089] Screening for antibodies binding to a particular epitope
(i.e., those binding to the same epitope) can be done using methods
routine in the art such as, e.g., without limitation, alanine
scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443-463),
peptide cleavage analysis, epitope excision, epitope extraction,
chemical modification of antigens (see Prot. Sci. 9 (2000)
487-496), and cross-blocking (see "Antibodies", Harlow and Lane
(Cold Spring Harbor Press, Cold Spring Harb., N.Y.).
[0090] Antigen Structure-based Antibody Profiling (ASAP), also
known as Modification-Assisted Profiling (MAP), allows to bin a
multitude of monoclonal antibodies specifically binding to an
antigen based on the binding profile of each of the antibodies from
the multitude to chemically or enzymatically modified antigen
surfaces (see, e.g., US 2004/0101920). The antibodies in each bin
bind to the same epitope which may be a unique epitope either
distinctly different from or partially overlapping with epitope
represented by another bin.
[0091] Also competitive binding can be used to easily determine
whether an antibody binds to the same epitope as, or competes for
binding with, a reference antibody. For example, an "antibody that
binds to the same epitope" as a reference antibody refers to an
antibody that blocks binding of the reference antibody to its
antigen in a competition assay by 50% or more, and conversely, the
reference antibody blocks binding of the antibody to its antigen in
a competition assay by 50% or more. Also for example, to determine
if an antibody binds to the same epitope as a reference antibody,
the reference antibody is allowed to bind to the antigen under
saturating conditions. After removal of the excess of the reference
antibody, the ability of an antibody in question to bind to the
antigen is assessed. If the antibody in question is able to bind to
the antigen after saturation binding of the reference antibody, it
can be concluded that the antibody in question binds to a different
epitope than the reference antibody. But, if the antibody in
question is not able to bind to the antigen after saturation
binding of the reference antibody, then the antibody in question
may bind to the same epitope as the epitope bound by the reference
antibody. To confirm whether the antibody in question binds to the
same epitope or is just hampered from binding by steric reasons
routine experimentation can be used (e.g., peptide mutation and
binding analyses using ELISA, RIA, surface plasmon resonance, flow
cytometry or any other quantitative or qualitative antibody-binding
assay available in the art). This assay should be carried out in
two set-ups, i.e. with both of the antibodies being the saturating
antibody. If, in both set-ups, only the first (saturating) antibody
is capable of binding to the antigen, then it can be concluded that
the antibody in question and the reference antibody compete for
binding to the antigen.
[0092] In some aspects, two antibodies are deemed to bind to the
same or an overlapping epitope if a 1-, 5-, 10-, 20- or 100-fold
excess of one antibody inhibits binding of the other by at least
50%, at least 75%, at least 90% or even 99% or more as measured in
a competitive binding assay (see, e.g., Junghans et al., Cancer
Res. 50 (1990) 1495-1502).
[0093] In some aspects, two antibodies are deemed to bind to the
same epitope if essentially all amino acid mutations in the antigen
that reduce or eliminate binding of one antibody also reduce or
eliminate binding of the other. Two antibodies are deemed to have
"overlapping epitopes" if only a subset of the amino acid mutations
that reduce or eliminate binding of one antibody reduce or
eliminate binding of the other.
[0094] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0095] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. In certain aspects, the antibody is of the IgG.sub.1
isotype. In certain aspects, the antibody is of the IgG.sub.1
isotype with the P329G, L234A and L235A mutation to reduce
Fc-region effector function. In other aspects, the antibody is of
the IgG.sub.2 isotype. In certain aspects, the antibody is of the
IgG.sub.4 isotype with the S228P mutation in the hinge region to
improve stability of IgG.sub.4 antibody. The heavy chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The light chain of an antibody may be assigned to one
of two types, called kappa (.kappa.) and lambda (.lamda.), based on
the amino acid sequence of its constant domain.
[0096] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g., B cell receptor); and B cell activation.
[0097] An "effective amount" of an agent, e.g., a pharmaceutical
composition, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0098] The term "tandem Fab" refers to an antibody comprising two
Fab fragments connected via a peptide linker/tether. In some
embodiments, a tandem Fab may comprise one Fab fragment and one
cross-Fab fragment, connected by a peptide linker/tether.
[0099] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one aspect, a human IgG heavy
chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, antibodies produced
by host cells may undergo post-translational cleavage of one or
more, particularly one or two, amino acids from the C-terminus of
the heavy chain. Therefore an antibody produced by a host cell by
expression of a specific nucleic acid molecule encoding a
full-length heavy chain may include the full-length heavy chain, or
it may include a cleaved variant of the full-length heavy chain.
This may be the case where the final two C-terminal amino acids of
the heavy chain are glycine (G446) and lysine (K447, numbering
according to EU index). Therefore, the C-terminal lysine (Lys447),
or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc
region may or may not be present. In one aspect, a heavy chain
including an Fc region as specified herein, comprised in an
antibody according to the invention, comprises an additional
C-terminal glycine-lysine dipeptide (G446 and K447, numbering
according to EU index). In one aspect, a heavy chain including an
Fc region as specified herein, comprised in an antibody according
to the invention, comprises an additional C-terminal glycine
residue (G446, numbering according to EU index). Unless otherwise
specified herein, numbering of amino acid residues in the Fc region
or constant region is according to the EU numbering system, also
called the EU index, as described in Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991.
[0100] "Framework" or "FR" refers to variable domain residues other
than complementary determining regions (CDRs). The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the CDR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.
[0101] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0102] The terms "host cell", "host cell line", and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells", which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0103] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0104] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one aspect,
for the VL, the subgroup is subgroup kappa I as in Kabat et al.,
supra. In one aspect, for the VH, the subgroup is subgroup III as
in Kabat et al., supra.
[0105] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human CDRs and amino acid
residues from human FRs. In certain aspects, a humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDRs
correspond to those of a non-human antibody, and all or
substantially all of the FRs correspond to those of a human
antibody. A humanized antibody optionally may comprise at least a
portion of an antibody constant region derived from a human
antibody. A "humanized form" of an antibody, e.g., a non-human
antibody, refers to an antibody that has undergone
humanization.
[0106] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and which determine antigen binding
specificity, for example "complementarity determining regions"
("CDRs").
[0107] Generally, antibodies comprise six CDRs: three in the VH
(CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2,
CDR-L3). Exemplary CDRs herein include:
[0108] (a) hypervariable loops occurring at amino acid residues
26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and
96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
[0109] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56
(L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)); and
[0110] (c) antigen contacts occurring at amino acid residues 27c-36
(L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101
(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).
[0111] Unless otherwise indicated, the CDRs are determined
according to Kabat et al., supra. One of skill in the art will
understand that the CDR designations can also be determined
according to Chothia, supra, McCallum, supra, or any other
scientifically accepted nomenclature system. Instead of the above,
the sequence of CDR-H1 as described herein may extend from Kabat26
to Kabat35, e.g., for the Pb-DOTAM binding variable domain.
[0112] In one aspect, CDR residues comprise those identified in the
sequence tables or elsewhere in the specification.
[0113] Unless otherwise indicated, HVR/CDR residues and other
residues in the variable domain (e.g., FR residues) are numbered
herein according to Kabat et al., supra.
[0114] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0115] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain aspects, the individual or subject is a
human.
[0116] Molecules as described herein may be "isolated". An
"isolated" antibody is one which has been separated from a
component of its natural environment. In some aspects, an antibody
is purified to greater than 95% or 99% purity as determined by, for
example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing
(IEF), capillary electrophoresis) or chromatographic (e.g., ion
exchange or reverse phase HPLC) methods. For a review of methods
for assessment of antibody purity, see, e.g., Flatman et al., J.
Chromatogr. B 848:79-87 (2007).
[0117] The term "nucleic acid molecule" or "polynucleotide"
includes any compound and/or substance that comprises a polymer of
nucleotides. Each nucleotide is composed of a base, specifically a
purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine
(A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or
ribose), and a phosphate group. Often, the nucleic acid molecule is
described by the sequence of bases, whereby said bases represent
the primary structure (linear structure) of a nucleic acid
molecule. The sequence of bases is typically represented from 5' to
3'. Herein, the term nucleic acid molecule encompasses
deoxyribonucleic acid (DNA) including e.g., complementary DNA
(cDNA) and genomic DNA, ribonucleic acid (RNA), in particular
messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed
polymers comprising two or more of these molecules. The nucleic
acid molecule may be linear or circular. In addition, the term
nucleic acid molecule includes both, sense and antisense strands,
as well as single stranded and double stranded forms. Moreover, the
herein described nucleic acid molecule can contain naturally
occurring or non-naturally occurring nucleotides. Examples of
non-naturally occurring nucleotides include modified nucleotide
bases with derivatized sugars or phosphate backbone linkages or
chemically modified residues. Nucleic acid molecules also encompass
DNA and RNA molecules which are suitable as a vector for direct
expression of an antibody of the invention in vitro and/or in vivo,
e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g.,
mRNA) vectors, can be unmodified or modified. For example, mRNA can
be chemically modified to enhance the stability of the RNA vector
and/or expression of the encoded molecule so that mRNA can be
injected into a subject to generate the antibody in vivo (see e.g.,
Stadler et al, Nature Medicine 2017, published online 12 Jun. 2017,
doi:10.1038/nm.4356 or EP 2 101 823 B1).
[0118] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0119] "Isolated nucleic acid encoding an antibody" refers to one
or more nucleic acid molecules encoding antibody heavy and light
chains (or fragments thereof), including such nucleic acid
molecule(s) in a single vector or separate vectors, and such
nucleic acid molecule(s) present at one or more locations in a host
cell.
[0120] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies in accordance with the present invention
may be made by a variety of techniques, including but not limited
to the hybridoma method, recombinant DNA methods, phage-display
methods, and methods utilizing transgenic animals containing all or
part of the human immunoglobulin loci, such methods and other
exemplary methods for making monoclonal antibodies being described
herein.
[0121] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
composition.
[0122] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable domain (VH), also
called a variable heavy domain or a heavy chain variable region,
followed by three constant heavy domains (CH1, CH2, and CH3).
Similarly, from N- to C-terminus, each light chain has a variable
domain (VL), also called a variable light domain or a light chain
variable region, followed by a constant light (CL) domain.
[0123] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0124] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity for
the purposes of the alignment. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
Clustal W, Megalign (DNASTAR) software or the FASTA program
package. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. Alternatively, the percent identity values can be
generated using the sequence comparison computer program ALIGN-2.
The ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc., and the source code has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087 and is described in WO 2001/007611.
[0125] Unless otherwise indicated, for purposes herein, percent
amino acid sequence identity values are generated using the
ggsearch program of the FASTA package version 36.3.8c or later with
a BLOSUM50 comparison matrix. The FASTA program package was
authored by W. R. Pearson and D. J. Lipman (1988), "Improved Tools
for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson
(1996) "Effective protein sequence comparison" Meth. Enzymol.
266:227-258; and Pearson et. al. (1997) Genomics 46:24-36 and is
publicly available from
www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.
ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server
accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be
used to compare the sequences, using the ggsearch (global
protein:protein) program and default options (BLOSUM50; open: -10;
ext: -2; Ktup=2) to ensure a global, rather than local, alignment
is performed. Percent amino acid identity is given in the output
alignment header.
[0126] The term "pharmaceutical composition" or "pharmaceutical
formulation" refers to a preparation which is in such form as to
permit the biological activity of an active ingredient contained
therein to be effective, and which contains no additional
components which are unacceptably toxic to a subject to which the
pharmaceutical composition would be administered.
[0127] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical composition or formulation, other
than an active ingredient, which is nontoxic to a subject. A
pharmaceutically acceptable carrier includes, but is not limited
to, a buffer, excipient, stabilizer, or preservative.
[0128] A reference to a target antigen as used herein, refers to
any native target antigen from any vertebrate source, including
mammals such as primates (e.g., humans) and rodents (e.g., mice and
rats), unless otherwise indicated. The term encompasses
"full-length", unprocessed target antigen as well as any form of
target antigen that results from processing in the cell. The term
also encompasses naturally occurring variants of the target
antigen, e.g., splice variants or allelic variants. For instance,
the target antigen CEA may have the amino acid sequence of human
CEA, in particular Carcinoembryonic antigen-related cell adhesion
molecule 5 (CEACAM5), which is shown in UniProt (www.uniprot.org)
accession no. P06731 (version 119), or NCBI (www.ncbi.nlm.nih.gov/)
RefSeq NP_004354.2. Another example of a target antigen is
Fibroblast Activation Protein (FAP). The amino acid sequence of
human FAP is shown in UniProt (www.uniprot.org) accession no.
Q12884 (version 149), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq
NP_004451.2. Another example of a target antigen is GPRC5D (see
UniProt no. Q9NZD1 (version 115); NCBI RefSeq no. NP_061124.1 for
the human sequence).
[0129] The terms "split antibody", "split antibodies", "single
domain split antibodies" or "SPLIT PRIT" as referred to herein mean
that the VH and VL domain which together form an antigen binding
site capable of binding to the radiolabelled compound are split
between two antibodies, and not present as part of the same
antibody (before assembly in vivo). "CEA-targeted SPLIT PRIT"
refers to a split antibody targeting CEA. The term "SPLIT PRIT" may
also be used interchangeably with the term "TA-split-DOTAM-VH/VL"
(e.g., where "TA" or target antigen is CEA, FAP or GPRC5D). The
term "CEA-targeted SPLIT PRIT" may be used interchangeably with the
term "CEA-split-DOTAM-VH/VL".
[0130] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of a disease
in the individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some aspects, antibodies of the
invention are used to delay development of a disease or to slow the
progression of a disease.
[0131] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
complementary determining regions (CDRs). (See, e.g., Kindt et al.
Kuby Immunology, 6.sup.th ed., W. H. Freeman and Co., page 91
(2007)) A single VH or VL domain may be sufficient to confer
antigen-binding specificity. Furthermore, antibodies that bind a
particular antigen may be isolated using a VH or VL domain from an
antibody that binds the antigen to screen a library of
complementary VL or VH domains, respectively. See, e.g., Portolano
et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature
352:624-628 (1991).
[0132] The term "vector", as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
[0133] The terms "Pb" or "lead" as used herein include ions
thereof, e.g., Pb(II). References to other metals also include ions
thereof. Thus, the skilled reader understands that, for example,
the terms lead, Pb, .sup.212Pb or .sup.203Pb are intended to
encompass ionic forms of the element, in particular, Pb(II).
II. Compositions and Methods
[0134] In one aspect, the invention is based, in part, on a set of
antibodies comprising a first and a second antibody, wherein each
antibody can bind to an antigen on a target cell, but wherein a
functional antigen binding site for an effector agent is formed
only when the first and second antibodies are associated with each
other. Antibodies of the invention are useful, e.g., for methods of
pre-targeted immunotherapy and/or for pre-targeted imaging. In
preferred aspects the methods eliminate a step of administering a
clearing agent or blocking agent.
A. Target Antigens
[0135] The antigen expressed on the surface of the target cell is
also termed herein the "target antigen".
[0136] Insofar as the invention relates to treatment methods and to
products for use therein, it is applicable to any condition that is
treatable by cytotoxic activity targeted to cells of the patient,
e.g., diseased cells. Thus, the target cell is any cell against
which it is desired to target cytotoxicity, e.g., any diseased
cell. The treatment is preferably of a tumour or cancer. However,
the applicability of the invention is not limited to tumours and
cancers. For example, the treatment may also be of viral infection
(by targeting infected cells) or T-cell driven autoimmune disease
(by targeting T cells). Immunotoxins directed against viral
antigens expressed on the surface of infected cells have been
investigated for a variety of viral infections such as HIV, rabies
and EBV. Cai and Berger 2011 Antiviral Research 90(3):143-50 used
an immunotoxin containing PE38 for targeted killing of cells
infected with Kaposi's sarcoma-associated herpesvirus. In addition,
Resimmune.RTM. (A-dmDT390-bisFv (UCHT1)) selectively kills human
malignant T cells and transiently depletes normal T cell and is
considered to have potential for the treatment of T-cell driven
autoimmune diseases such as multiple sclerosis and
graft-versus-host disease, as well as T cell blood cancers for
which it is undergoing clinical trials. Likewise, methods of the
invention may be applicable to any cell type for which radioimaging
is desirable, including but not limited to cancer or tumour
cells.
[0137] Thus, suitable target antigens may include cancer cell
antigens, viral antigens or microbial antigens.
[0138] The antigens are usually normal cell surface antigens which
are either over-expressed or expressed at abnormal times. Ideally
the target antigen is expressed only on diseased cells (such as
tumour cells), however this is rarely observed in practice. As a
result, target antigens are usually selected on the basis of
differential expression between diseased and healthy tissue.
[0139] The cell surface marker or target antigen can be, for
example, a tumour-associated antigen.
[0140] The term "tumour-associated antigen" or "tumour specific
antigen" as used herein refers to any molecule (e.g., protein,
peptide, lipid, carbohydrate, etc.) solely or predominantly
expressed or over-expressed by tumour cells and/or cancer cells, or
by other cells of the stroma of the tumour such as
cancer-associated fibroblasts, such that the antigen is associated
with the tumour(s) and/or cancer(s). The tumour-associated antigen
can additionally be expressed by normal, non-tumour, or
non-cancerous cells. However, in such cases, the expression of the
tumour-associated antigen by normal, non-tumour, or non-cancerous
cells is not as robust as the expression by tumour or cancer cells.
In this regard, the tumour or cancer cells can over-express the
antigen or express the antigen at a significantly higher level, as
compared to the expression of the antigen by normal, non-tumour, or
non-cancerous cells. Also, the tumour-associated antigen can
additionally be expressed by cells of a different state of
development or maturation. For instance, the tumour-associated
antigen can be additionally expressed by cells of the embryonic or
foetal stage, which cells are not normally found in an adult host.
Alternatively, the tumour-associated antigen can be additionally
expressed by stem cells or precursor cells, which cells are not
normally found in an adult host.
[0141] The tumour-associated antigen can be an antigen expressed by
any cell of any cancer or tumour, including the cancers and tumours
described herein. The tumour-associated antigen may be a
tumour-associated antigen of only one type of cancer or tumour,
such that the tumour-associated antigen is associated with or
characteristic of only one type of cancer or tumour. Alternatively,
the tumour-associated antigen may be a tumour-associated antigen
(e.g., may be characteristic) of more than one type of cancer or
tumour. For example, the tumour-associated antigen may be expressed
by both breast and prostate cancer cells and not expressed at all
by normal, non-tumour, or non-cancer cells.
[0142] Exemplary tumour-associated antigens to which the antibodies
of the invention may bind include, but are not limited to, mucin 1
(MUC1; tumour-associated epithelial mucin), preferentially
expressed antigen of melanoma (PRAME), carcinoembryonic antigen
(CEA), prostate specific membrane antigen (PSMA), PSCA, EpCAM,
Trop2 (trophoblast-2, also known as EGP-1), granulocyte-macrophage
colony-stimulating factor receptor (GM-CSFR), CD56, human epidermal
growth factor receptor 2 (HER2/neu) (also known as erbB-2), CDS,
CD7, tyrosinase related protein (TRP) I, and TRP2. In another
embodiment, the tumour antigen may be selected from the group
consisting of cluster of differentiation (CD) 19, CD20, CD21, CD22,
CD25, CD30, CD33 (sialic acid binding Ig-like lectin 3, myeloid
cell surface antigen), CD79b, CD123 (interleukin 3 receptor alpha),
transferrin receptor, EGF receptor, mesothelin, cadherin, Lewis Y,
Glypican-3, FAP (fibroblast activation protein alpha), GPRC5D (G
Protein-Coupled Receptor Class C Group 5 Member D), PSMA (prostate
specific membrane antigen), CA9=CAIX (carbonic anhydrase IX), L1
CAM (neural cell adhesion molecule L 1), endosialin, HER3
(activated conformation of epidermal growth factor receptor family
member 3), Alkl/BMP9 complex (anaplastic lymphoma kinase 1/bone
morphogenetic protein 9), TPBG=5T4 (trophoblast glycoprotein), ROR1
(receptor tyrosine kinase-like surface antigen), HER1 (activated
conformation of epidermal growth factor receptor), and CLL1 (C-type
lectin domain family 12, member A). Mesothelin is expressed in,
e.g., ovarian cancer, mesothelioma, non-small cell lung cancer,
lung adenocarcinoma, fallopian tube cancer, head and neck cancer,
cervical cancer, and pancreatic cancer. CD22 is expressed in, e.g.,
hairy cell leukaemia, chronic lymphocytic leukaemia (CLL),
prolymphocytic leukaemia (PLL), non-Hodgkin's lymphoma, small
lymphocytic lymphoma (SLL), and acute lymphatic leukaemia (ALL).
CD25 is expressed in, e.g., leukemias and lymphomas, including
hairy cell leukaemia and Hodgkin's lymphoma. Lewis Y antigen is
expressed in, e.g., bladder cancer, breast cancer, ovarian cancer,
colorectal cancer, esophageal cancer, gastric cancer, lung cancer,
and pancreatic cancer. CD33 is expressed in, e.g., acute myeloid
leukaemia (AML), chronic myelomonocytic leukaemia (CML), and
myeloproliferative disorders.
[0143] Exemplary antibodies that specifically bind to
tumour-associated antigens include, but are not limited to,
antibodies against the transferrin receptor (e.g., HB21 and
variants thereof), antibodies against CD22 (e.g., RFB4 and variants
thereof), antibodies against CD25 (e.g., anti-Tac and variants
thereof), antibodies against mesothelin (e.g., SS 1, MORAb-009, SS,
HN1, HN2, MN, MB, and variants thereof) and antibodies against
Lewis Y antigen (e.g., B3 and variants thereof). In this regard,
the targeting moiety (cell-binding agent) may be an antibody
selected from the group consisting of B3, RFB4, SS, SS1, MN, MB,
HN1, HN2, HB21, and MORAb-009, and antigen binding portions
thereof. Further exemplary targeting moieties suitable for use in
the inventive chimeric molecules are disclosed e.g., in U.S. Pat.
No. 5,242,824 (anti-transferrin receptor); 5,846,535 (anti-CD25);
5,889,157 (anti-Lewis Y); 5,981,726 (anti-Lewis Y); 5,990,296
(anti-Lewis Y); U.S. Pat. No. 7,081,518 (anti-mesothelin);
7,355,012 (anti-CD22 and anti-CD25); U.S. Pat. No. 7,368,110
(anti-mesothelin); 7,470,775 (anti-CD30); 7,521,054 (anti-CD25);
and 7,541,034 (anti-CD22); U.S. Patent Application Publication
2007/0189962 (anti-CD22); Frankel et al., Clin. Cancer Res., 6:
326-334 (2000), and Kreitman et al., AAPS Journal, 8(3): E532-E551
(2006), each of which is incorporated herein by reference.
[0144] Further antibodies have been raised to target specific
tumour related antigens including: Cripto, CD30, CD19, CD33,
Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD22
(Siglec2), CD33 (Siglec3), CD79, CD138, PSCA, PSMA (prostate
specific membrane antigen), BCMA, CD20, CD70, E-selectin, EphB2,
Melanotransferin, Muc16 and TMEFF2. Any of these, or
antigen-binding fragments thereof, may be useful in the present
invention, i.e., may be incorporated into the antibodies described
herein.
[0145] In some embodiments of the present invention, it may be
preferred that the tumour-associated antigen is carcinoembryonic
antigen (CEA).
[0146] CEA is advantageous in the context of the present invention
because it is relatively slowly internalized, and thus a high
percentage of the antibody will remain available on the surface of
the cell after initial treatment, for binding to the radionuclide.
Other low internalizing targets/tumour associated antigens may also
be preferred. Other examples of tumour-associated antigen include
CD20 or HER2. In still further embodiments, the target may be EGP-1
(epithelial glycoprotein-1, also known as trophoblast-2),
colon-specific antigen-p (CSAp) or a pancreatic mucin MUC1. See for
instance Goldenberg et al 2012 (Theranostics 2(5)), which is
incorporated herein by reference. This reference also describes
antibodies such as Mu-9 binding to CSAp (see also Sharkey et al
Cancer Res. 2003; 63: 354-63), hPAM4 binding to MUC1 (see also Gold
et al Cancer Res. 2008: 68: 4819-26), valtuzumab binding to CD20
(see also Sharkey et al Cancer Res. 2008; 68: 5282-90) and hRS7
which binds to EGP-1 (see also Cubas et al Biochim Biophys Acta
2009; 1796: 309-14). Any of these or antigen-binding portions
thereof may be useful in the present invention, i.e., may be
incorporated into the antibodies described herein. One example of
an antibody that has been raised against CEA is T84.66 (as shown in
NCBI Acc No: CAA36980 for the heavy chain and CAA36979 for the
light chain, or as shown in SEQ ID NO 317 and 318 of WO2016/075278)
and humanized and chimeric versions thereof, such as T84.66-LCHA as
described in WO2016/075278 A1 and/or WO2017/055389. Another example
is CH1A1a, an anti-CEA antibody as described in WO2012/117002 and
WO2014/131712, and CEA hMN-14 (see also U.S. Pat. Nos. 6,676,924
and 5,874,540). Another anti-CEA antibody is A5B7 as described in
M. J. Banfield et al, Proteins 1997, 29(2), 161-171. Humanized
antibodies derived from murine antibody A5B7 have been disclosed in
WO 92/01059 and WO 2007/071422. See also co-pending application
PCT/EP2020/067582. An example of a humanized version of A5B7 is
A5H1EL1 (G54A). A further exemplary antibody against CEA is MFE23
and the humanized versions thereof described in U.S. Pat. No.
7,626,011 and/or co-pending application PCT/EP2020/067582. A still
further example of an antibody against CEA is 28A9. Any of these or
an antigen binding fragment thereof may be useful to form a
CEA-binding moiety in the present invention.
[0147] FAP (fibroblast activation protein alpha) or GPRC5D (G
Protein-Coupled Receptor Class C Group 5 Member D) may also be
preferred in some embodiments. FAP is an established target for
imaging and therapy, due to its broad expression in the
microenvironment of a number of tumor types, e.g. pancreas, breast,
and lung cancer (Lindner, T., Loktev, A., Giesel, F. et al.
Targeting of activated fibroblasts for imaging and therapy. EJNMMI
radiopharm. chem. 4, 16 (2019)). SPLIT PRIT using
FAP-split-DOTAM-VH/VL antibodies would thus be expected to generate
specific accumulation of .sup.212Pb-DOTAM on activated
cancer-associated fibroblasts. Consequently, the emitted alpha
radiation would be expected to negatively affect the immune
suppression of FAP-expressing malignant tumors, in addition to a
limited direct tumor-killing effect on adjacent tumor cells.
G-protein coupled receptor family C group 5 member D (GPRC5D) is
overexpressed on multiple myeloma plasma cells (Atamaniuk J, Gleiss
A, Porpaczy E, Kainz B, Grunt T W, Raderer M, et al. Overexpression
of G protein-coupled receptor 5D in the bone marrow is associated
with poor prognosis in patients with multiple myeloma. Eur J Clin
Invest. 2012; 42:953-60), with established SC (subcutaneous) in
vivo models reflecting the expression found in multiple myeloma
patients, e.g. OPM-2 and NCI-H929 (Kodama T, Kochi Y, Nakai W,
Mizuno H, Baba T, Habu K, et al. Anti-GPRC5D/CD3 bispecific
T-cell-redirecting antibody for the treatment of multiple myeloma.
Mol Cancer Ther. (2019) 18:1555-64). We therefore expect SPLIT PRIT
using GPRC5D-split-DOTAM-VH/VL antibodies to generate
tumor-specific accumulation of 212Pb-DOTAM followed by
radiation-induced tumor cell death.
[0148] In some embodiments, the antibodies of the invention may
bind specifically to the target antigen (e.g., any of the target
antigens discussed herein). In some embodiments, they may bind with
a dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-7M or less, e.g. from 10.sup.-7 to
10.sup.-13, 10.sup.-8 M or less, e.g. from 10.sup.-8 M to
10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0149] The first and second antibody each bind to the same target
antigen, which can be termed "antigen A" (i.e., they have binding
specificity for the same target antigen). They may each having
binding specificity for the same epitope on antigen A.
Alternatively, the first antibody may bind to a first epitope on
antigen A and the second antibody may bind to a different, second
epitope on antigen A. For instance, in one embodiment, one of the
antibodies may bind to the T84.66 epitope of CEA and the other may
bind to the A5B7 epitope of CEA.
[0150] In some embodiments, one or both of the first and/or second
antibodies may be biparatopic for antigen A--i.e., each of the
individual antibodies may bind to two different epitopes of antigen
A. The first antibody may comprise a first and a second binding
site, which bind to a first and a second epitope of antigen A
respectively, wherein the first and second epitopes are different
from each other. Alternatively or additionally, the second antibody
may comprise a first and a second binding site, which bind to a
first and a second epitope of antigen A, wherein the first and
second epitopes are different from each other. In some embodiments,
one or both of the epitopes bound by the first antibody may be
different from one or both of the epitopes bound by the second
antibody. In other embodiments, the two epitopes bound by the first
antibody may be the same as the two epitopes bound by the second
antibody.
B. Radiolabelled Compounds
[0151] According to the present invention, association of the first
and second antibodies forms a functional binding site for an
effector molecule. Effector molecules according to the present
invention are radiolabelled compounds which comprise a
radioisotope, e.g., are a radiolabelled hapten.
[0152] In some embodiments, the effector molecule may comprise a
chelated radioisotope.
[0153] In some embodiments, the functional binding site for the
effector molecule may bind to a chelate comprising the chelator and
the radioisotope. In other embodiments, the antibody may bind to a
moiety which is conjugated to the chelated radioisotope, for
instance, histamine-succinyl-glycine (HSG), digoxigenin, biotin or
caffeine
[0154] The chelator may be, for example, a multidentate molecule
such as an aminopolycarboxylic acid or an aminopolythiocarboxylic
acid, or a salt or functional variant thereof. The chelator may be,
for example, bidentate or tridentate or tetradentate. Examples of
suitable metal chelators include molecules comprising EDTA
(Ethylenediaminetetraacetic acid, or a salt form such as
CaNa.sub.2EDTA), DTPA (Diethylenetriamine Pentaacetic Acid), DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA
(2,2',2''-(1,4,7-Triazanonane-1,4,7-triyl)triacetic acid), IDA
(Iminodiacetic acid), MIDA ((Methylimino)diacetic acid), TTHA
(3,6,9,12-Tetrakis(carboxymethyl)-3,6,9,12-tetra-azatetradecanedioic
acid), TETA
(2,2',2'',2'''-(1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetrayl)tetraa-
cetic acid), DOTAM
(1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane),
HEHA
(1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic
acid, available from Macrocyclics, Inc., Plano, Tex.), NTA
(nitrilotriacetic acid) EDDHA (ethylenediamine-N,
N'-bis(2-hydroxyphenylacetic acid), BAL (2,3,-dimercaptopropanol),
DMSA (2,3-dimercaptosuccinic acid), DMPS
(2,3-dimercapto-1-propanesulfonic acid), D-penicillamine
(B-dimethylcysteine), MAG.sub.3 (mercaptoacetyltriglycine), Hynic
(6-hydrazinopridine-3-carboxylic acid),
p-isothiocyanatobenzyl-desferrioxamine (e.g., labelled with
zirkonium for imaging), and salts or functional
variants/derivatives thereof capable of chelating the metal. In
some embodiments, it may be preferred that the chelator is DOTA or
DOTAM or a salt or functional variant/derivative thereof capable of
chelating the metal. Thus, the chelator may be or may comprise DOTA
or DOTAM with a radioisotope chelated thereto.
[0155] The effector molecule may comprise or consist of functional
variants or derivatives of the chelators above, together with the
radionuclide. Suitable variants/derivatives have a structure that
differs to a certain limited extent and retain the ability to
function as a chelator (i.e. retains sufficient activity to be used
for one or more of the purposes described herein). Functional
variants/derivatives may also include a chelator as described above
conjugated to one or more additional moieties or substituents,
including, a small molecule, a polypeptide or a carbohydrate. This
attachment may occur via one of the constituent carbons, for
example in a backbone portion of the chelator. A suitable
substituent can be, for example, a hydrocarbon group such as alkyl,
alkenyl, aryl or alkynyl; a hydroxy group; an alcohol group; a
halogen atom; a nitro group; a cyano group; a sulfonyl group; a
thiol group; an amine group; an oxo group; a carboxy group; a
thiocarboxy group; a carbonyl group; an amide group; an ester
group; or a heterocycle including heteroaryl groups. The
substituent may be, for example, one of those defined for group
"R.sup.1" below. A small molecule can be, for example, a dye (such
as Alexa 647 or Alexa 488), biotin or a biotin moiety, or a phenyl
or benzyl moiety. A polypeptide may be, for example, an oligo
peptide, e.g., an oligopeptide of two or three amino acids.
Exemplary carbohydrates include dextran, linear or branched
polymers or co-polymers (e.g. polyalkylene, poly(ethylene-lysine),
polymethacrylate, polyamino acids, poly- or oligosaccharides,
dendrimers). Derivatives may also include multimers of the chelator
compounds in which compounds as set out above are linked through a
linker moiety. Derivatives may also include functional fragments of
the above compounds, which retain the ability to chelate the metal
ion.
[0156] Particular examples of derivatives include benzyl-EDTA and
hydroxyethyl-thiourido-benzyl EDTA, DOTA-benzene (e.g.,
(S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic
acid), DOTA-biotin, and DOTA-TyrLys-DOTA.
[0157] In some embodiments of the present invention, the functional
binding site formed by association of the first and second antibody
binds to a metal chelate comprising DOTAM and a metal, e.g., lead
(Pb). As mentioned above, "DOTAM" has the chemical name:
[0158]
1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane,
which is a compound of the following formula:
##STR00001##
[0159] The present invention may in certain aspects and embodiments
also make use of functional variants or derivatives of DOTAM
incorporating a metal ion. Suitable variants/derivatives of DOTAM
have a structure that differs to a certain limited extent from the
structure of DOTAM and retain the ability to function (i.e. retains
sufficient activity to be used for one or more of the purposes
described herein). In such aspects and embodiments, the DOTAM or
functional variant/derivative of DOTAM may be one of the active
variants disclosed in WO 2010/099536. Suitable functional
variants/derivatives may be a compound of the following
formula:
##STR00002##
or a pharmaceutically acceptable salt thereof; wherein
[0160] R.sup.N is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl,
C.sub.3-7 cycloalkyl-C.sub.1-4 alkyl, C.sub.2-7 heterocycloalkyl,
C.sub.2-7 heterocycloalkyl-C.sub.1-4 alkyl, phenyl,
phenyl-C.sub.1-4-alkyl, C.sub.1-7 heteroaryl, and
C.sub.1-7heteroaryl-C.sub.1-4-alkyl; wherein C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl are
each optionally substituted by 1, 2, 3, or 4 independently selected
R.sup.w groups; and wherein said C.sub.3-7 cycloalkyl, C.sub.3-7
cycloalkyl-C.sub.1-4alkyl, C.sub.2-7 heterocycloalkyl, C.sub.2-7
heterocycloalkyl-C.sub.1-4 alkyl, phenyl, phenyl-C.sub.1-4-alkyl,
C.sub.1-7 heteroaryl, and C.sub.1-7 heteroaryl-C.sub.1-4-alkyl are
each optionally substituted by 1, 2, 3, or 4 independently selected
R.sup.x groups;
[0161] L.sup.1 is independently C.sub.1-6 alkylene, C.sub.1-6
alkenylene, or C.sub.1-6 alkynylene, each of which is optionally
substituted by 1, 2, or 3 groups independently selected R.sup.1
groups;
[0162] L.sup.2 is C.sub.2-4 straight chain alkylene, which is
optionally substituted by an independently selected R.sup.1 group;
and which is optionally substituted by 1, 2, 3, or 4 groups
independently selected from C.sub.1-4 alkyl and or C.sub.1-4
haloalkyl;
[0163] R.sup.1 is independently selected from
D.sup.1-D.sup.2-D.sup.3, halogen, cyano, nitro, hydroxyl, C.sub.1-6
alkoxy, C.sub.1-6 haloalkoxy, C.sub.1-6 alkylthio, C.sub.1-6
alkylsulfinyl, C.sub.1-6 alkylsulfonyl, amino, C.sub.1-6
alkylamino, di-C.sub.1-6 alkylamino, C.sub.1-4 alkylcarbonyl,
carboxy, C.sub.1-6 alkoxycarbonyl, C.sub.1-6 alkylcarbonylamino,
di-C.sub.1-6 alkylcarbonylamino, C.sub.1-6 alkoxycarbonylamino,
C.sub.1-6 alkoxycarbonyl-(C.sub.1-6 alkyl)amino, carbamyl,
C.sub.1-6 alkylcarbamyl, and di-C.sub.1-6 alkylcarbamyl;
[0164] each D.sup.1 is independently selected from C.sub.6-10
aryl-C.sub.1-4 alkyl, C.sub.1-9 heteroaryl-C.sub.1-4 alkyl,
C.sub.3-10 cycloalkyl-C.sub.1-4 alkyl, C.sub.2-9
heterocycloalkyl-C.sub.1-4 alkyl, C.sub.1-8 alkylene, C.sub.1-8
alkenylene, and C.sub.1-8 alkynylene; wherein said C.sub.1-8
alkylene, C.sub.1-8 alkenylene, and C.sub.1-8 alkynylene are
optionally substituted by 1, 2, 3, or 4 independently selected
R.sup.4 groups; and wherein said C.sub.6-10 aryl-C.sub.1-4 alkyl,
C.sub.1-9 heteroaryl-C.sub.1-4 alkyl, C.sub.3-10
cycloalkyl-C.sub.1-4 alkyl, C.sub.2-9 heterocycloalkyl-C.sub.1-4
alkyl are each optionally substituted by 1, 2, 3, or 4
independently selected R.sup.5 groups;
[0165] each D.sup.2 is independently absent or C.sub.1-20 straight
chain alkylene, wherein from 1 to 6 non-adjacent methylene groups
of said C.sub.1-20 straight chain alkylene are each optionally
replaced by an independently selected --D.sup.4-- moiety, provided
that at least one methylene unit in said C.sub.1-20 straight chain
alkylene is not optionally replaced by a --D.sup.4-- moiety;
wherein said C.sub.1-20 straight chain alkylene is optionally
substituted by one or more groups independently selected from
halogen, cyano, nitro, hydroxyl, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkoxy, amino, C.sub.1-4
alkylamino, di-C.sub.1-4 alkylamino, C.sub.1-4 alkylcarbonyl,
carboxy, C.sub.1-4 alkoxycarbonyl, C.sub.1-4 alkylcarbonylamino,
di-C.sub.1-4 alkylcarbonylamino, C.sub.1-4 alkoxycarbonylamino,
C.sub.1-4 alkoxycarbonyl-(C.sub.1-4 alkyl)amino, carbamyl,
C.sub.1-4 alkylcarbamyl, and di-C.sub.1-4 alkylcarbamyl;
[0166] each D.sup.3 is independently selected from H, halogen,
cyano, nitro, hydroxyl, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-14 cycloalkyl,
C.sub.3-14 cycloalkyl-C.sub.1-4 alkyl, C.sub.2-14 heterocycloalkyl,
C.sub.2-14 heterocycloalkyl-C.sub.1-4 alkyl, C.sub.6-14 aryl,
C.sub.6-14 aryl-C.sub.1-4 alkyl, C.sub.1-13 heteroaryl, C.sub.1-13
heteroaryl-C.sub.1-4 alkyl; wherein said C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl are each optionally
substituted by 1, 2, 3, or 4 independently selected R.sup.6 groups;
and wherein said C.sub.3-14 cycloalkyl, C.sub.3-14
cycloalkyl-C.sub.1-4 alkyl, C.sub.2-14 heterocycloalkyl, C.sub.2-14
heterocycloalkyl-C.sub.1-4 alkyl, C.sub.6-14 aryl, C.sub.6-14
aryl-C.sub.1-4 alkyl, C.sub.1-13 heteroaryl, C.sub.1-13
heteroaryl-C.sub.1-4 alkyl are each optionally substituted by 1, 2,
3 or 4 independently selected R.sup.7 groups;
[0167] each D.sup.4 is independently selected from --O--, --S--,
--NR.sup.aC(.dbd.O)--, --NR.sup.aC(.dbd.S)--,
--NR.sup.bC(.dbd.O)NR.sup.c--, --NR.sup.bC(.dbd.S)NR.sup.c--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, --S(.dbd.O)NR.sup.a--,
--C(.dbd.O)--, --C(.dbd.S)--, --C(.dbd.O)O--,
--OC(.dbd.O)NR.sup.a--, --OC(.dbd.S)NR.sup.a--, --NR.sup.a--,
--NR.sup.bS(.dbd.O)NR.sup.c--, and
NR.sup.bS(.dbd.O).sub.2NR.sup.O--;
[0168] each R.sup.4 and R.sup.6 is independently selected from
halogen, cyano, nitro, hydroxyl, C.sub.1-4 alkoxy, C.sub.1-4
haloalkoxy, C.sub.1-4 alkylthio, C.sub.1-4 alkylsulfinyl, C.sub.1-4
alkylsulfonyl, amino, C.sub.1-4 alkylamino, di-C.sub.1-4
alkylamino, C.sub.1-4 alkylcarbonyl, carboxy, C.sub.1-4
alkoxycarbonyl, C.sub.1-4 alkylcarbonylamino, di-C.sub.1-4
alkylcarbonylamino, C.sub.1-4 alkoxycarbonylamino, C.sub.1-4
alkoxycarbonyl-(C.sub.1-4 alkyl)amino, carbamyl, C.sub.1-4
alkylcarbamyl, and di-C.sub.1-4 alkylcarbamyl;
[0169] each R.sup.5 is independently selected from halogen, cyano,
cyanate, isothiocyanate, nitro, hydroxyl, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.1-4 alkoxy, C.sub.1-4
haloalkoxy, C.sub.1-4 alkylthio, C.sub.1-4 alkylsulfinyl, C.sub.1-4
alkylsulfonyl, amino, C.sub.1-4 alkylamino, di-C.sub.1-4
alkylamino, C.sub.1-4 alkylcarbonyl, carboxy, C.sub.1-4
alkoxycarbonyl, C.sub.1-4 alkylcarbonylamino, di-C.sub.1-4
alkylcarbonylamino, C.sub.1-4 alkoxycarbonylamino, C.sub.1-4
alkoxycarbonyl-(C.sub.1-4 alkyl)amino, carbamyl, C.sub.1-4
alkylcarbamyl, and di-C.sub.1-4 alkylcarbamyl;
[0170] each R.sup.7 is independently selected from halogen, cyano,
nitro, hydroxyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.3-7 cycloalkyl, C.sub.3-7 cycloalkyl-C.sub.1-4
alkyl, C.sub.2-7 heterocycloalkyl, C.sub.2-7
heterocycloalkyl-C.sub.1-4 alkyl, phenyl, phenyl-C.sub.1-4 alkyl,
C.sub.1-7 heteroaryl, C.sub.1-7 heteroaryl-C.sub.1-4 alkyl,
--OR.sup.O, --SR.sup.O, --S(.dbd.O)R.sup.P,
--S(.dbd.O).sub.2R.sup.P, --S(.dbd.O)NR.sup.sR.sup.t,
--C(.dbd.O)R.sup.P, --C(.dbd.O)OR.sup.P,
--C(.dbd.O)NR.sup.sR.sup.t, --OC(.dbd.O)R.sup.P,
--OC(.dbd.O)NR.sup.sR.sup.t, --NR.sup.sR.sup.t,
--NR.sup.qC(.dbd.O)R.sup.r, --NR.sup.qC(.dbd.O)OR.sup.r,
--NR.sup.4C(.dbd.O)NR.sup.r, --NR.sup.9S(.dbd.O).sub.2W, and
--NR.sup.PS(.dbd.O).sub.2NR.sup.sR.sup.t; wherein said C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl are each optionally
substituted by 1, 2, 3, or 4 independently selected R' groups; and
wherein said C.sub.3-7 cycloalkyl, C.sub.3-7 cycloalkyl-C.sub.1-4
alkyl, C.sub.2-7 heterocycloalkyl, C.sub.2-7
heterocycloalkyl-C.sub.1-4 alkyl, phenyl, phenyl-C.sub.1-4 alkyl,
C.sub.1-7 heteroaryl, C.sub.1-7 heteroaryl-C.sub.1-4 alkyl are each
optionally substituted by 1, 2, 3, or 4 independently selected R''
groups;
[0171] each R.sup.a, R.sup.b, and R.sup.c is independently selected
from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl, C.sub.3-7
cycloalkyl-C.sub.1-4 alkyl, C.sub.2-7 heterocycloalkyl, C.sub.2-7
heterocycloalkyl-C.sub.1-4 alkyl, phenyl, phenyl-C.sub.1-4 alkyl,
C.sub.1-7 heteroaryl, C.sub.1-7 heteroaryl-C.sub.1-4 alkyl; wherein
said C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl are each optionally substituted by 1, 2, 3, or 4
independently selected R.sup.w groups; and wherein said C.sub.3-7
cycloalkyl, C.sub.3-7 cycloalkyl-C.sub.1-4 alkyl, C.sub.2-7
heterocycloalkyl, C.sub.2-7 heterocycloalkyl-C.sub.1-4 alkyl,
phenyl, phenyl-C.sub.1-4 alkyl, C.sub.1-7 heteroaryl, C.sub.1-7
heteroaryl-C.sub.1-4 alkyl are each optionally substituted by 1, 2,
3, or 4 independently selected R.sup.x groups;
[0172] each R.sup.o, R.sup.p, R.sup.q, R.sup.r, R.sup.s and R.sup.t
is independently selected from H, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7
cycloalkyl, C.sub.3-7 cycloalkyl-C.sub.1-4 alkyl, C.sub.2-7
heterocycloalkyl, C.sub.2-7 heterocycloalkyl-C.sub.1-4 alkyl,
phenyl, phenyl-C.sub.1-4 alkyl, C.sub.1-7 heteroaryl, C.sub.1-7
heteroaryl-C.sub.1-4 alkyl; wherein said C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl are each optionally
substituted by 1, 2, 3, or 4 independently selected RY groups; and
wherein said C.sub.3-7 cycloalkyl, C.sub.3-7 cycloalkyl-C.sub.1-4
alkyl, C.sub.2-7 heterocycloalkyl, C.sub.2-7
heterocycloalkyl-C.sub.1-4 alkyl, phenyl, phenyl-C.sub.1-4 alkyl,
C.sub.1-7 heteroaryl, C.sub.1-7 heteroaryl-C.sub.1-4 alkyl are each
optionally substituted by 1, 2, 3, or 4 independently selected
R.sup.z groups;
[0173] each R', R.sup.w and R.sup.y is independently selected from
hydroxyl, cyano, nitro, C.sub.1-4 alkoxy, C.sub.1-4 haloalkoxy,
amino, C.sub.1-4 alkylamino, and di-C.sub.1-4 alkylamino; and
[0174] each R'', R.sup.x, and R.sup.z is independently selected
from hydroxyl, halogen, cyano, nitro, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkoxy, amino, C.sub.1-4
alkylamino, and di-C.sub.1-4 alkylamino;
[0175] provided that the valency of each atom in the optionally
substituted moieties is not exceeded.
[0176] Suitably, the functional variants/derivatives of the above
formula have an affinity for an antibody of the present invention
which is comparable to or greater than that of DOTAM, and have a
binding strength for Pb which is comparable to or greater than that
of DOTAM ("affinity" being as measured by the dissociation
constant, as described above). For example, the dissociation
constant of the functional/variant derivative with the antibody of
the present invention or/Pb may be 1.1 times or less, 1.2 times or
less, 1.3 times or less, 1.4 times or less, 1.5 times or less, or 2
times or less than the dissociation constant of DOTAM with the same
antibody/Pb.
[0177] Each R.sup.N may be H, C.sub.1-6 alkyl, or C.sub.1-6
haloalkyl; preferably H, C.sub.1-4 alkyl, or C.sub.1-4 haloalkyl.
Most preferably, each R.sup.N is H.
[0178] For DOTAM variants, it is preferred that 1, 2, 3 or most
preferably each L.sup.2 is C.sub.2 alkylene. Advantageously, the
C.sub.2 alkylene variants of DOTAM can have particularly high
affinity for Pb. The optional substituents for L.sup.2 may be
R.sup.1, C.sub.1-4 alkyl, or C.sub.1-4 haloalkyl. Suitably, the
optional substituents for L.sup.2 may be C.sub.1-4 alkyl or
C.sub.1-4 haloalkyl.
[0179] Optionally, each L.sup.2 may be unsubstituted C.sub.2
alkylene --CH2CH2-.
[0180] Each L.sup.1 is preferably C.sub.1-4 alkylene, more
preferably C.sub.1 alkylene such as --CH2-.
[0181] The functional variant/derivative of DOTAM may be a compound
of the following formula:
##STR00003##
wherein each Z is independently R.sup.1 as defined above; p, q, r,
and s are 0, 1 or 2; and p+q+r+s is 1 or greater. Preferably, p, q,
r, and s are 0 or 1 and/or p+q+r+s is 1. For example, the compound
may have p+q+r+s=1, where Z is p-SCN-benzyl moiety--such a compound
is commercially available from Macrocyclics, Inc. (Plano,
Tex.).
[0182] Radionuclides useful in the invention may include
radioisotopes of metals, such as of lead (Pb), lutetium (Lu), or
yttrium (Y).
[0183] Radionuclides particularly useful in imaging applications
may be radionuclides that are gamma emitters. For instance, they
may be selected from .sup.203Pb or .sup.205Bi.
[0184] Radionuclides particularly useful in therapeutic
applications be radionuclides that are alpha or beta emitters. For
instance, they may be selected from .sup.212Pb, .sup.212Bi,
.sup.213Bi, .sup.90Y, .sup.177Lu, .sup.225Ac, .sup.211At,
.sup.227Th, .sup.223Ra.
[0185] In some embodiments, it may be preferred that DOTAM (or
salts or functional variants thereof) is chelated with Pb or Bi
such as one of the Pb or Bi radioisotopes listed above. It other
embodiments, it may be preferred that DOTA (or salts or functional
variants thereof) is chelated with Lu or Y such as one of the Lu or
Y radioisotopes listed above.
[0186] In some embodiments, methods and uses may comprise combined
methods of therapy and imaging, which make use of a mixture of
radioisotopes, e.g., a radioisotope suitable for therapy and a
radioisotope suitable for imaging. For instance, these may be
different radioisotopes of the same metal, chelated by the same
chelator. In one embodiment, the method may comprise administering
.sup.203Pb-DOTAM and .sup.212Pb-DOTAM as a mixture. In another
embodiment, the method may comprise a first cycle of dosimetry
using a gamma emitter such as .sup.203Pb or .sup.205Bi followed by
one or more rounds of treatment using an alpha or beta emitter such
as .sup.212Pb, .sup.212Bi, .sup.213Bi, .sup.90Y, .sup.177Lu,
.sup.225Ac, .sup.211At, .sup.227Th, or .sup.223Ra. Such methods are
described further below.
[0187] In some embodiments, the functional binding site formed by
association of the first and the second antibody may bind to a
Pb-DOTAM chelate.
[0188] In some embodiments, the functional binding site formed by
association of the first and the second antibody may specifically
bind to the radiolabelled compound. In some embodiments, it may
bind to the radiolabelled compound, such as the Pb-DOTAM chelate,
with a dissociation constant (Kd) to Pb-DOTAM and/or the target of
.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM,
.ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g.
10.sup.-7M or less, e.g. from 10.sup.-7 to 10.sup.-13, 10.sup.-8 M
or less, e.g. from 10.sup.-8 M to 10-13 M, e.g., from 10.sup.-9 M
to 10.sup.-13 M). It some embodiments it may be preferred that it
binds with a Kd value of the binding affinity of 100 pM, 50 pM, 20
pM, 10 pM, 5 pM, 1 pM or less, e.g., 0.9 pM or less, 0.8 pM or
less, 0.7 pM or less, 0.6 pM or less or 0.5 pM or less. For
instance, the functional binding site may bind the metal chelate
with a Kd of about 1 pM-1 nM, e.g., about 1-10 pM, 1-100 pM, 5-50
pM, 100-500 pM or 500 pM-1 nM.
C. Exemplary Antigen Binding Sites for DOTA
[0189] In one particular embodiment of the invention, the first and
second antibody associate to form a functional binding site for
DOTA (or a functional derivative or variant thereof), e.g., DOTA
chelated with Lu or Y (e.g., .sup.177Lu or .sup.90Y). For instance,
the functional binding site may bind the radiolabelled compound
with a Kd of about 1 pM-1 nM, e.g., about 1-10 pM, 1-100 pM, 5-50
pM, 100-500 pM or 500 pM-1 nM.
[0190] C825 is a known scFv with high affinity for DOTA-Bn
(S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic
acid) complexed with radiometals such as .sup.177Lu and .sup.90Y
(see for instance Cheal et al 2018, Theranostics 2018, and
WO2010099536, incorporated herein by reference). The CDR sequences
and the VL and VH sequences of C825 are provided herein. In one
embodiment, the heavy chain variable region forming part of the
antigen binding site for the radiolabelled compound may comprise at
least one, two or all three CDRs selected from (a) CDR-H1
comprising the amino acid sequence of 35; (b) CDR-H2 comprising the
amino acid sequence of 36; (c) CDR-H3 comprising the amino acid
sequence of 37. In an alternative embodiment, CDR-H1 may have the
sequence GFSLTDYGVH (SEQ ID NO.: 148). The light chain variable
region forming part of the binding site for the radiolabelled
compound may comprise at least one, two or all three CDRs selected
from (d) CDR-L1 comprising the amino acid sequence of 38; (e)
CDR-L2 comprising the amino acid sequence of 39; and (0 CDR-L3
comprising the amino acid sequence of 40.
[0191] In another embodiment, the heavy chain variable domain
forming part of the functional antigen binding site for the
radiolabelled compound (on the first antibody) comprises the amino
acid sequence of SEQ ID NO: 41, or a variant thereof comprising an
amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97,
98, or 99% identity to SEQ ID NO: 41. In certain embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but a binding site comprising that sequence retains the
ability to bind to DOTA complexed with Lu or Y, preferably with an
affinity as described herein. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:41. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the CDRs (i.e., in the FRs).
Optionally, the antibody comprises the VH sequence in SEQ ID NO:41,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:35 or the sequence GFSLTDYGVH (SEQ ID NO.: 148), (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:36, and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:37.
[0192] Optionally, the light chain variable domain forming part of
the functional antigen binding site for the radiolabelled compound
(on the second antibody) comprises an amino acid sequence of SEQ ID
NO: 42 or a variant thereof comprising an amino acid sequence
having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity
to SEQ ID NO: 42. In certain embodiments, a VL sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but a
binding site comprising that sequence retains the ability to bind
to DOTA complexed with Lu or Y, preferably with an affinity as
described herein. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID NO:
42. In certain embodiments, the substitutions, insertions, or
deletions occur in regions outside the CDRs (i.e., in the FRs).
Optionally, the antibody comprises the VL sequence in SEQ ID NO:42,
including post-translational modifications of that sequence. In a
particular embodiment, the VL comprises one, two or three CDRs
selected from (a) CDR-L1 comprising the amino acid sequence of SEQ
ID NO:38; (b) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:39; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:40.
[0193] Embodiments concerned with the heavy chain variable region
and the light chain variable region are explicitly contemplated in
combination. Thus, the functional antigen binding site may be
formed from a heavy chain variable region as defined above and a
light chain variable region as defined above, on the first and
second antibody respectively.
[0194] In any of the above embodiments, the light and heavy chain
variable regions forming the binding site for the DOTA complex may
be humanized. In one embodiment, the light and heavy chain variable
region comprise CDRs as in any of the above embodiments, and
further comprise an acceptor human framework, e.g. a human
immunoglobulin framework or a human consensus framework.
[0195] In some embodiments, the heavy chain variable domain may be
extended by one or more C-terminal residues such as one or more
C-terminal alanine residues, or one or more residues from the
N-terminus of the CH1 domain, as discussed further below.
D. Exemplary Antigen Binding Sites for DOTAM
[0196] In another particular embodiment of the invention, the first
and second antibody associate to form a functional antigen binding
site for a Pb-DOTAM chelate (Pb-DOTAM).
[0197] In certain embodiments, the functional antigen-binding site
that binds to Pb-DOTAM may have one or more of the following
properties: [0198] Binds specifically to Pb-DOTAM and to Bi-DOTAM;
[0199] Is selective for Pb-DOTAM (and optionally Bi-DOTAM) as
compared to other chelated metals, such as Cu-DOTAM; [0200] Binds
to Pb-DOTAM with a very high affinity; [0201] Binds to the same
epitope on Pb-DOTAM as antibodies described herein, e.g., PRIT-0213
or PRIT-0214 and/or has the same contact residues as said
antibodies.
[0202] Radioisotopes of Pb are useful in methods of diagnosis and
therapy. Particular radioisotopes of lead which may be of use in
the present invention include .sup.212Pb and .sup.203Pb.
[0203] Radionuclides which are .alpha.-particle emitters have the
potential for more specific tumour cell killing with less damage to
the surrounding tissue than .beta.-emitters because of the
combination of short path length and high linear energy transfer.
.sup.212Bi is an .alpha.-particle emitter but its short half-life
hampers its direct use. .sup.212Pb is the parental radionuclide of
.sup.212Bi and can serve as an in vivo generator of .sup.212Bi,
thereby effectively overcoming the short half-life of .sup.212Bi
(Yong and Brechbiel, Dalton Trans. 2001 Jun. 21; 40 (23)
6068-6076).
[0204] .sup.203Pb is useful as an imaging isotope. Thus, an
antibody bound to .sup.203Pb-DOTAM may have utility in
radioimmunoimaging (RIO.
[0205] Generally, radiometals are used in chelated form. In certain
aspects of the present invention, DOTAM is used as the chelating
agent. DOTAM is a stable chelator of Pb(II) (Yong and Brechbiel,
Dalton Trans. 2001 Jun. 21; 40 (23) 6068-6076; Chappell et al
Nuclear Medicine and Biology, Vol. 27, pp. 93-100, 2000). Thus,
DOTAM is particularly useful in conjunction with isotopes of lead
as discussed above, such as .sup.212Pb and .sup.203Pb.
[0206] In some embodiments, it may be preferred that the antibodies
bind Pb-DOTAM with a Kd value of the binding affinity of 100 pM, 50
pM, 20 pM, 10 pM, 5 pM, 1 pM or less, e.g, 0.9 pM or less, 0.8 pM
or less, 0.7 pM or less, 0.6 pM or less or 0.5 pM or less. For
instance, the functional binding site may bind the radiolabelled
compound with a Kd of about 1 pM-1 nM, e.g., about 1-10 pM, 1-100
pM, 5-50 pM, 100-500 pM or 500 pM-1 nM.
[0207] In certain embodiment, the antibodies additionally bind to
Bi chelated by DOTAM. In some embodiments, it may be preferred that
the antibodies bind Bi-DOTAM (i.e., a chelate comprising DOTAM
complexed with bismuth, also termed herein a "Bi-DOTAM chelate")
with a Kd value of the binding affinity of 1 nM, 500 pM, 200 pM,
100 pM, 50 pM, 10 pM or less, e.g., 9 pM, 8 pM, 7 pM, 6 pM, 5 pM or
less. For instance, the functional binding site may bind a metal
chelate with a Kd of about 1 pM-1 nM, e.g., about 1-10 pM, 1-100
pM, 5-50 pM, 100-500 pM or 500 pM-1 nM.
[0208] In some embodiments, the antibodies may bind to Bi-DOTAM and
to Pb-DOTAM with a similar affinity. For instance, it may be
preferred that the ratio of affinity, e.g., the ratio of Kd values,
for Bi-DOTAM/Pb-DOTAM is in the range of 0.1-10, for example
1-10.
[0209] In one embodiment, the heavy chain variable region forming
part of the antigen binding site for Pb-DOTAM may comprise at least
one, two or all three CDRs selected from (a) CDR-H1 comprising the
amino acid sequence of GFSLSTYSMS (SEQ ID NO:1); (b) CDR-H2
comprising the amino acid sequence of FIGSRGDTYYASWAKG (SEQ ID
NO:2); (c) CDR-H3 comprising the amino acid sequence of
ERDPYGGGAYPPHL (SEQ ID NO:3). The light chain variable region
forming part of the binding site for Pb-DOTAM may comprise at least
one, two or all three CDRs selected from (d) CDR-L1 comprising the
amino acid sequence of QSSHSVYSDNDLA (SEQ ID NO:4); (e) CDR-L2
comprising the amino acid sequence of QASKLAS (SEQ ID NO:5); and
(f) CDR-L3 comprising the amino acid sequence of LGGYDDESDTYG (SEQ
ID NO:6).
[0210] In some embodiments, the antibodies may comprise one or more
of CDR-H1, CDR-H2 and/or CDR-H3, or one or more of CDR-L1, CDR-L2
and/or CDR-L3, having substitutions as compared to the amino acid
sequences of SEQ ID NOs: 1-6, respectively, e.g., 1, 2 or 3
substitutions.
[0211] In some embodiments, antibodies may share the same contact
residues as the described herein: e.g., these residues may be
invariant. These residues may include the following: [0212] a) in
heavy chain CDR2: Phe50, Asp56 and/or Tyr58, and optionally also
Gly52 and/or Arg 54; [0213] b) in heavy chain CDR3: Glu95, Arg96,
Asp97, Pro98, Tyr99, Ala100C and/or Tyr100D and optionally also
Pro100E; [0214] c) in light chain CDR1: Tyr28 and/or Asp32; [0215]
d) in light chain CDR3: Gly91, Tyr92, Asp93, Thr95c and/or Tyr96;
[0216] e) in light chain CDR2: optionally G1n50; [0217] all
numbered according to Kabat.
[0218] For example, in some embodiments, CDR-H2 may comprise the
amino acid sequence FIGSRGDTYYASWAKG (SEQ ID NO:2), or a variant
thereof having up to 1, 2, or 3 substitutions in SEQ ID NO: 2,
wherein these substitutions do not include Phe50, Asp56 and/or
Tyr58, and optionally also do not include Gly52 and/or Arg 54, all
numbered according to Kabat.
[0219] In some embodiments, CDR-H2 may be substituted at one or
more positions as shown below. Here and in the substitution tables
that follow, substitutions are based on the germline residues
(underlined) or by amino acids which theoretically sterically fit
and also occur in the crystallized repertoire at the site. In some
embodiments, the residues as mentioned above may be fixed and other
residues may be substituted according to the table below: in other
embodiments, substitutions of any residue may be made according to
the table below.
TABLE-US-00001 WolfGuy Kabat AA Substitution 251 50 F Y, H 252 51 I
253 52 G 254 53 S A, G, T, I, N 288 54 R A, D, G, N, S, T, F, Y 289
55 G D, S, Y, T, A, N, R, V 290 56 D 291 57 T K, I, A, P, S 292 58
Y F, W, H 293 59 Y N, F, H, L, S 294 60 A G, N, S, T 295 61 S A, G,
N, Q, T 296 62 W K, P, S, A, T, D, N, R, Q 297 63 A F, L, V, M, I
298 64 K N, Q, R, E 299 65 G S, T, D, N, A
[0220] Optionally, CDR-H3 may comprise the amino acid sequence
ERDPYGGGAYPPHL (SEQ ID NO:3), or a variant thereof having up to 1,
2, or 3 substitutions in SEQ ID NO: 3, wherein these substitutions
do not include Glu95, Arg96, Asp97, Pro98, and optionally also do
not include Ala100C, Tyr100D, and/or Pro100E and/or optionally also
do not include Tyr99. For instance, in some embodiments the
substitutions do not include Glu95, Arg96, Asp97, Pro98, Tyr99
Ala100C and Tyr100D.
[0221] In certain embodiments, CDR-H3 may be substituted at one or
more positions as shown below. In some embodiments, the residues as
mentioned above may be fixed and other residues may be substituted
according to the table below: in other embodiments, substitutions
of any residue may be made according to the table below.
TABLE-US-00002 WolfGuy Kabat AA Substitution 351 95 E 352 96 R K, E
353 97 D 354 98 P 355 99 Y F, G, S, T, D 356 100 G 392 100A G 393
100B G 394 100C A S, T 395 100D Y F 396 100E P 397 100F P 398 101 H
A, T, V, D 399 102 L Y, V, I, H, F
[0222] Optionally, CDR-L1 may comprise the amino acid sequence
QSSHSVYSDNDLA (SEQ ID NO:4) or a variant thereof having up to 1, 2,
or 3 substitutions in SEQ ID NO: 4, wherein these substitutions do
not include Tyr28 and/or Asp32 (Kabat numbering).
[0223] In certain embodiments, CDR-L1 may be substituted at one or
more positions as shown below. Again, in some embodiments, the
residues as mentioned above may be fixed and other residues may be
substituted according to the table below: in other embodiments,
substitutions of any residue may be made according to the table
below.
TABLE-US-00003 WolfGuy Kabat AA Substitution 551 24 Q R, K 552 25 S
A, G 554 26 S T 555 27 H Q, S, R, K 556 27A S Q 557 27B V I, D, N
561 28 Y F 562 29 S T, V 571 30 D R, S, N, G 572 31 N K 597 32 D
598 33 L I, V, M 599 34 A S
[0224] Optionally, CDR-L3 may comprise the amino acid sequence
LGGYDDESDTYG (SEQ ID NO:6) or a variant thereof having up to 1, 2,
or 3 substitutions in SEQ ID NO: 6, wherein these substitutions do
not include Gly91, Tyr92, Asp93, Thr95c and/or Tyr96 (Kabat).
[0225] In certain embodiments, CDR-L3 may be substituted at the
following positions as shown below. (Since most residues are
solvent exposed and without antigen contacts, many substitutions
are conceivable). Again, in some embodiments, the residues as
mentioned above may be fixed and other residues may be substituted
according to the table below: in other embodiments, substitutions
of any residue may be made according to the table below.
TABLE-US-00004 WolfGuy Kabat AA Substitution 751 89 L A, V, Q 752
90 G A 753 91 G 754 92 Y A, D, E, F, G, H, I, K, L, N, Q, R, S, T,
V 755 93 D A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 756 94
D A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 794 95 E A, D,
F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 795 95A S A, F, G, H,
I, K, L, M, N, Q, R, T, V, W, Y 796 95B D A, E, F, G, H, I, L, M,
N, Q, S, T, V, W, Y 797 95C T S 798 96 Y F, H, R 799 97 G A, E, I,
K, L, M, N, Q, S, T, V
[0226] The antibody may further comprise CDR-H1 or CDR-L2,
optionally having the sequence of SEQ ID NO: 1 or SEQ ID NO: 5
respectively, or a variant thereof having at least 1, 2 or 3
substitutions relative thereto, optionally conservative
substitutions.
[0227] Thus, the heavy chain variable domain forming part of the
antigen binding site for Pb-DOTAM may comprise at least:
a) heavy chain CDR2 comprising the amino acid sequence
FIGSRGDTYYASWAKG (SEQ ID NO:2), or a variant thereof having up to
1, 2, or 3 substitutions in SEQ ID NO: 2, wherein these
substitutions do not include Phe50, Asp56 and/or Tyr58, and
optionally also do not include Gly52 and/or Arg54; b) heavy chain
CDR3 comprising the amino acid sequence ERDPYGGGAYPPHL (SEQ ID
NO:3), or a variant thereof having up to 1, 2, or 3 substitutions
in SEQ ID NO: 3, wherein these substitutions do not include Glu95,
Arg96, Asp97, Pro98, and optionally also do not include Ala100C,
Tyr100D, and/or Pro100E and/or optionally also do not include
Tyr99.
[0228] In some embodiments, the heavy chain variable domain
additionally includes a heavy chain CDR1 which is optionally:
c) a heavy chain CDR1 comprising the amino acid sequence GFSLSTYSMS
(SEQ ID NO:1) or a variant thereof having up to 1, 2, or 3
substitutions in SEQ ID NO: 1.
[0229] In some embodiments, the heavy chain variable domain
additionally includes a C-terminal alanine (e.g. Ala114 according
Kabat numbering system) to avoid the binding of pre-existing
antibodies recognizing the free VH region. As reported in Holland M
C et al J. Clin Immunol (2013), a free C-terminus appears to be
important for binding of HAVH (human anti-VH domain) autoantibodies
to VH domain antibodies, since HAVH autoantibodies do not bind to
intact IgG or IgG fragments (fAb or modified VH molecules)
containing the same VH framework sequences, or to VK domain
antibodies. Cordy J C et al Clinical and Experimental Immunology
(2015) notes the existence of a cryptic epitope at the C-terminal
epitope of VH dAbs, which is not naturally accessible to HAVH
antibodies in full IgG molecules.
[0230] Thus, where the antibody comprises a free VH region (not
fused to any other domain at its C-terminus), the sequence may be
extended by one or more C-terminal residue. The extension may
prevent the binding of antibodies recognizing the free VH region.
For instance, the extension may be by 1-10 residues, e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9 or 10 residues. In one embodiment, the VH sequence
may be extended by one or more C-terminal alanine residues. The VH
sequence may also be extended by an N-terminal portion of the CH1
domain, e.g., by 1-10 residues from the N-terminus of the CH1
domain, e.g., from the human IgG1 CH1 domain. (The first ten
residues of the human IgG1 CH1 domain are ASTKGPSVFP (SEQ ID NO.:
149), and so in one embodiment, from 1-10 residues may be taken
from the N-terminus of this sequence). For instance, in one
embodiment, the peptide sequence AST (corresponding to the first 3
residues of the IgG1 CH1 domain) is added to the C-terminus of the
VH region.
[0231] In another embodiment, the light chain variable domain
forming part of the antigen binding site for Pb-DOTAM comprises at
least: [0232] d) light chain CDR1 comprising the amino acid
sequence QSSHSVYSDNDLA (SEQ ID NO:4) or a variant thereof having up
to 1, 2, or 3 substitutions in SEQ ID NO: 4, wherein these
substitutions do not include Tyr28 and Asp32; [0233] e) light chain
CDR3 comprising the amino acid sequence LGGYDDESDTYG (SEQ ID NO:6)
or a variant thereof having up to 1, 2, or 3 substitutions in SEQ
ID NO: 6, wherein these substitutions do not include Gly91, Tyr92,
Asp93, Thr95c and Tyr96.
[0234] In some embodiments, the light chain variable domain
additionally includes a light chain CDR2 which is optionally:
[0235] f) a light chain CDR2 comprising the amino acid sequence
QASKLAS (SEQ ID NO: 5) or a variant thereof having at least 1, 2 or
3 substitutions in SEQ ID NO: 5, optionally not including
Gln50.
[0236] In any embodiments of the present invention which include
variants of a sequence comprising the CDRs as set out above (e.g.,
of a variable domain), the protein may be invariant in one or more
of the CDR residues as set out above.
[0237] Optionally, the heavy chain variable domain forming part of
the functional antigen binding site for Pb-DOTAM (on the first
antibody) comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 7 and SEQ ID NO 9, or a variant thereof
comprising an amino acid sequence having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 7 or SEQ ID NO:
9. In certain embodiments, a VH sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but a binding site
comprising that sequence retains the ability to bind to Pb-DOTAM,
preferably with an affinity as described herein. The VH sequence
may retain the invariant residues as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO: 7 or SEQ ID NO 9. In certain
embodiments, substitutions, insertions, or deletions occur in
regions outside the CDRs (i.e., in the FRs). Optionally, the
antibody comprises the VH sequence in SEQ ID NO:7 or SEQ ID NO: 9,
including post-translational modifications of that sequence,
optionally with a C-terminal Ala. In a particular embodiment, the
VH comprises one, two or three CDRs selected from: (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:1, (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:3.
[0238] In some embodiments, as mentioned above, in some variants
SEQ ID NO: 7 or 9 may be extended by one or more additional
C-terminal residues, e.g., by one or more alanine residues,
optionally a single alanine residue. Thus, for instance, in one
specific variant, the sequence of SEQ ID NO: 7 may be extended to
be:
TABLE-US-00005 (SEQ ID NO.: 150)
VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKA
LEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDP
VDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA
[0239] In other embodiments, the extension may be by an N-terminal
portion of the CH1 domain as described above, e.g., by 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the extension may be by the
peptide sequence AST.
[0240] Optionally, the light chain variable domain forming part of
the functional antigen binding site for Pb-DOTAM (on the second
antibody) comprises an amino acid sequence of SEQ ID NO: 8, or a
variant thereof comprising an amino acid sequence having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:
8. In certain embodiments, a VL sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-Pb-DOTAM
binding site comprising that sequence retains the ability to bind
to Pb-DOTAM, preferably with an affinity as described herein. The
VL sequence may retain the invariant residues as set out above. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO:8. In certain
embodiments, the substitutions, insertions, or deletions occur in
regions outside the CDRs (i.e., in the FRs). Optionally, the
anti-Pb-DOTAM antibody comprises the VL sequence in SEQ ID NO:8,
including post-translational modifications of that sequence. In a
particular embodiment, the VL comprises one, two or three CDRs
selected from (a) CDR-L1 comprising the amino acid sequence of SEQ
ID NO:4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:6.
[0241] Embodiments concerned with the heavy chain variable region
and the light chain variable region are explicitly contemplated in
combination. Thus, the functional antigen binding site for Pb-DOTAM
may be formed from a heavy chain variable region as defined above
and a light chain variable region as defined above, on the first
and second antibody respectively.
[0242] Optionally, the antigen binding site specific for the
Pb-DOTAM chelate may be formed from a heavy chain variable domain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 7 or SEQ ID NO: 9, or a variant thereof as
defined above (including a variant with a C-terminal extension as
discussed above), and a light chain variable domain comprising an
amino acid sequence of SEQ ID NO: 8, or a variant thereof as
defined above. For example, the antigen binding site specific for
the Pb-DOTAM chelate may comprise a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO: 7 or a variant
thereof, and a light chain variable domain comprising the amino
acid sequence of SEQ ID NO: 8 or a variant thereof, including
post-translational modifications of those sequences. In another
embodiment, it may comprise a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO: 9 or a variant
thereof (including a variant with a C-terminal extension as
discussed above) and a light chain variable domain comprising the
amino acid sequence of SEQ ID NO: 8 or a variant thereof, including
post-translational modifications of those sequences.
[0243] In any of the above embodiments, the light and heavy chain
variable regions forming the anti-Pb-DOTAM binding site may be
humanized. In one embodiment, the light and heavy chain variable
region comprise CDRs as in any of the above embodiments, and
further comprise an acceptor human framework, e.g. a human
immunoglobulin framework or a human consensus framework. In another
embodiment, the light and/or heavy chain variable regions comprise
CDRs as in any of the above embodiments, and further comprises
framework regions derived from vk 1 39 and/or vh 2 26. For vk 1 39,
in some embodiments there may be no back mutations. For vh 2 26,
the germline Ala49 residue may be backmutated to Gly49.
E. Exemplary Antigen Binding Sites for CEA
[0244] In another particular embodiment of the present invention,
which may be combined with the embodiments discussed above, the
target antigen bound by the first and second antibody may be CEA
(carcinoembryonic antigen). Antibodies that have been raised
against CEA include T84.66 and humanized and chimeric versions
thereof, such as T84.66-LCHA as described in WO2016/075278 A1
and/or WO2017/055389, CH1Ala, an anti-CEA antibody as described in
WO2012/117002 and WO2014/131712, and CEA hMN-14 or labetuzimab
(e.g., as described in U.S. Pat. Nos. 6,676,924 and 5,874,540).
Another exemplary antibody against CEA is A5B7 (e.g., as described
in M. J. Banfield et al, Proteins 1997, 29(2), 161-171), or a
humanized antibody derived from murine A5B7 as described in WO
92/01059 and WO 2007/071422. See also co-pending application
PCT/EP2020/067582. An example of a humanized version of A5B7 is
A5H1EL1 (G54A). A further exemplary antibody against CEA is MFE23
and the humanized versions thereof described in U.S. Pat. No.
7,626,011 and/or co-pending application PCT/EP2020/067582. A still
further example of an anti-CEA antibody is 28A9. Any of these or
antigen binding fragments thereof may be used to form a CEA-binding
moiety in the present invention.
[0245] Optionally, the antigen-binding site which binds to CEA may
bind with a Kd value of 1 nM or less, 500 pM or less, 200 pM or
less, or 100 pM or less for monovalent binding.
[0246] In some embodiments, the first and/or second antibody may
bind to the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope,
the T84.66 epitope or the 28A9 epitope of CEA.
[0247] In some embodiments, at least one of the first and second
antibodies binds to a CEA epitope which is not present on soluble
CEA (sCEA). Soluble CEA is a part of the CEA molecule which is
cleaved by GPI phospholipase and released into the blood. An
example of an epitope not found on soluble CEA is the CH1A1A
epitope. Optionally, one of the first and/or second antibody binds
to an epitope which is not present on soluble CEA, and the other
binds to an epitope which is present on soluble CEA.
[0248] The epitope for CH1A1a and its parent murine antibody PR1A3
is described in WO2012/117002A1 and Durbin H. et al., Proc. Natl.
Scad. Sci. USA, 91:4313-4317, 1994. An antibody which binds to the
CH1A1a epitope binds to a conformational epitope within the B3
domain and the GPI anchor of the CEA molecule. In one aspect, the
antibody binds to the same epitope as the CH1A1a antibody having
the VH of SEQ ID NO: 25 and VL of SEQ ID NO 26 herein. The A5B7
epitope is described in co-pending application PCT/EP2020/067582.
An antibody which binds to the A5B7 epitope binds to the A2 domain
of CEA, i.e., to the domain comprising the amino acids of SEQ ID
NO: 154:
TABLE-US-00006 (SEQ ID NO: 154)
PKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPV
SPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVI LN.
In one aspect, the antibody binds to the same epitope as the A5B7
antibody having the VH of SEQ ID NO: 49 and VL of SEQ ID NO: 50
herein.
[0249] In one aspect, the antibody binds to the same epitope as the
T84.66 described in WO2016/075278. The antibody may bind to the
same epitope as the antibody having the VH of SEQ ID NO: 17 and VL
of SEQ ID NO:18 herein.
[0250] The MFE23 epitope is described in co-pending application
PCT/EP2020/067582. An antibody which binds to the MFE23 epitope
binds to the A1 domain of CEA, i.e., to the domain comprising the
amino acids of SEQ ID NO: 155:
TABLE-US-00007 (SEQ ID NO: 155)
PKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPV
SPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVI LN.
In one aspect, the antibody may bind to the same epitope as an
antibody having the VH domain of SEQ ID NO: 167 and the VL domain
of SEQ ID NO: 168 herein.
[0251] In some embodiments, the first and/or second antibody may
bind to the same CEA-epitope as an antibody provided herein, e.g.,
P1AD8749, P1AD8592, P1AE4956, P1AE4957, P1AF0709, P1AF0298,
P1AF0710 or P1AF0711.
[0252] In some embodiments, the first and the second antibody bind
the same epitope of CEA as each other. Thus, for example, the first
and the second antibody may both bind to the CH1A1a epitope, the
A5B7 epitope, the MFE23 epitope, the T84.66 epitope or the 28A9
epitope.
[0253] In some embodiments, both the first and second antibody may
have CEA binding sequences (i.e., CDRs and/or VH/VL domains) from
CH1A1A; or, the first and the second antibody may both have CEA
binding sequences from A5B7 or a humanized version thereof; or, the
first and the second antibody may both have CEA binding sequences
from T84.66 or a humanized version thereof; or the first and the
second antibody may both have CEA binding sequences from MFE23 or a
humanized version thereof; or the first and second antibody may
both have CEA binding sequences from 28A9 or a humanized version
thereof. Exemplary sequences are disclosed herein.
[0254] In other embodiments, the first and the second antibodies
bind to different epitopes of CEA. Thus, for example, i) one
antibody may bind the CH1A1A epitope and the other may bind the
A5B7 epitope, the T84.66 epitope, the MFE23 epitope or the 28A9
epitope; ii) one antibody may bind the A5B7 epitope and the other
may bind the CH1A1A epitope, T84.66 epitope, MFE23 epitope or 28A9
epitope; iii) one antibody may bind the MFE23 epitope and the other
may bind the CH epitope, A5B7 epitope, T84.66 epitope or 28A9
epitope; iv) one antibody may bind the T84.66 epitope and the other
may bind the CH1A1A epitope, A5B7 epitope, MFE23 epitope or 28A9
epitope; or v) one antibody may bind the 28A9 epitope and the other
may bind the CH1A1a epitope, the A5B7 epitope, the MFE23 epitope,
or the T84.66 epitope.
[0255] In some embodiments, i) one antibody may have CEA binding
sequences (i.e., CDRs or VH/VL domains) from CH1A1A and the other
may have CEA binding sequences from A5B7 or a humanized version
thereof, from T84.66 or a humanized version thereof, from MFE23 or
a humanized version thereof, or from 28A9 or a humanized version
thereof; ii) one antibody may have CEA binding sequences from A5B7
or a humanized version thereof and the other may have CEA binding
sequences from CH1A1A, from T84.66 or a humanized version thereof,
from MFE23 or a humanized version thereof, or from 28A9 or a
humanized version thereof; iii) one antibody may have CEA binding
sequences from MFE23 or a humanized version thereof and the other
may have CEA binding sequences from CH1A1A, from A5B7 or a
humanized version thereof, from T84.66 or a humanized version
thereof, or from 28A9 or a humanized version thereof; iv) one
antibody may have CEA binding sequences from T84.66 or a humanized
version thereof and the other may have CEA binding sequences from
CH1A1A, from A5B7 or a humanized version thereof, from MFE23 or a
humanized version thereof, or from 28A9 or a humanized version; v)
one antibody may have CEA-binding sequences from 28A9 or a
humanized version thereof and the other may have CEA binding
sequences from CH1A1A, from A5B7 or a humanized version thereof,
from T84.66 or a humanized version thereof, or from MFE23 or a
humanized version thereof.
[0256] In one particular embodiment, one antibody may bind the
CH1A1A epitope and the other may bind the A5B7 epitope. The first
antibody may have CEA binding sequences from the antibody CH and
the second antibody may have CEA binding sequences from A5B7
(including a humanized version thereof); or, the first antibody may
have CEA binding sequences from the antibody A5B7 (including a
humanized version thereof) and the second antibody may have CEA
binding sequences from CH1A1A.
[0257] In another particular embodiment, one antibody may bind the
CH epitope and the other may bind the T84.66 epitope. The first
antibody may have CEA binding sequences from the antibody CH and
the second antibody may have CEA binding sequences from T84.66
(including a humanized version thereof); or, the first antibody may
have CEA binding sequences from the antibody T84.66 (including a
humanized version thereof) and the second antibody may have CEA
binding sequences from CH1A1A. In some embodiments, a first
antibody may bind the T84.66 epitope and/or have an antigen binding
site as described in (i) below, and the second antibody may bind
the CH1A1A epitope and/or have an antigen binding site as described
in (ii) below.
[0258] Exemplary CEA-binding sequences i)-v) are disclosed below.
These provide examples of CEA-binding sequences from i) T84.66, ii)
CH1A1A, iii) A5B7, iv) 28A9 and v) MFE23 (or from humanized
versions thereof).
i). In one embodiment, the antigen-binding site which binds to CEA
may comprise at least one, two, three, four, five, or six CDRs
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:12; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:13; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:16.
[0259] Optionally, the antigen-binding site which binds to CEA may
comprise at least one, at least two, or all three VH CDR sequences
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:12; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:13.
[0260] Optionally, the antigen-binding site which binds to CEA
comprises at least one, at least two, or all three VL CDRs
sequences selected from (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:14; (b) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:15; and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:16.
[0261] Optionally, the antigen-binding site which binds to CEA
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:11, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:12, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:13; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:14, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:15, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:16.
[0262] In another aspect, the antigen-binding site which binds to
CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:11; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:12; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:13; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:14; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:15; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:16.
[0263] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-CEA antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0264] In another embodiment, the antigen-binding site which binds
to CEA comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:17. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to CEA, preferably with the affinity as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:17. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the antigen-binding site
which binds to CEA comprises the VH sequence in SEQ ID NO:17,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:11, (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:12, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:13.
[0265] In another embodiment, the antigen-binding site which binds
to CEA comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:18. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but the antigen-binding site comprising
that sequence retains the ability to bind to CEA, preferably with
the affinity set out above. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:18. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the antigen-binding site for CEA comprises
the VL sequence in SEQ ID NO:18, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three CDRs selected from (a) CDR-L1
comprising the amino acid sequence of SEQ ID NO:14; (b) CDR-L2
comprising the amino acid sequence of SEQ ID NO:15; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:16.
[0266] In another embodiment, the antigen-binding site which binds
to CEA comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:17 and SEQ ID NO:18, respectively, including
post-translational modifications of those sequences.
ii). In further particular embodiment, the antigen-binding site
which binds to CEA may comprise at least one, two, three, four,
five, or six CDRs selected from (a) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:19; (b) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:20; (c) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:21; (d) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23; and (f) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0267] Optionally, the antigen-binding site which binds to CEA may
comprise at least one, at least two, or all three VH CDR sequences
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:21.
[0268] Optionally, the antigen-binding site which binds to CEA
comprises at least one, at least two, or all three VL CDRs
sequences selected from (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22; (b) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23; and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0269] Optionally, the antigen-binding site which binds to CEA
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:21; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:22, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:23, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0270] In another aspect, the antigen-binding site which binds to
CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:24.
[0271] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-CEA antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0272] In another embodiment, the antigen-binding site which binds
to CEA comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:25. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to CEA, preferably with the affinity as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:25. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the antigen-binding site
which binds to CEA comprises the VH sequence in SEQ ID NO:25,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:19, (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:20, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:21.
[0273] In another embodiment, the antigen-binding site which binds
to CEA comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:26. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but the antigen-binding site comprising
that sequence retains the ability to bind to CEA, preferably with
the affinity set out above. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:26. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the antigen-binding site for CEA comprises
the VL sequence in SEQ ID NO:26, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three CDRs selected from (a) CDR-L1
comprising the amino acid sequence of SEQ ID NO:22; (b) CDR-L2
comprising the amino acid sequence of SEQ ID NO:23; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:24.
[0274] In another embodiment, the antigen-binding site which binds
to CEA comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:25 and SEQ ID NO:26, respectively, including
post-translational modifications of those sequences.
iii) In further particular embodiment, the antigen-binding site
which binds to CEA may comprise at least one, two, three, four,
five, or six CDRs selected from (a) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:43; (b) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:44; (c) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:45; (d) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:46; (e) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:47; and (f) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:48. In some embodiments, CDR-H1 may have the
sequence GFTFTDYYMN (SEQ ID NO.: 151).
[0275] Optionally, the antigen-binding site which binds to CEA may
comprise at least one, at least two, or all three VH CDR sequences
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:44; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:45. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN
(SEQ ID NO.: 151).
[0276] Optionally, the antigen-binding site which binds to CEA
comprises at least one, at least two, or all three VL CDRs
sequences selected from (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:46; (b) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:47; and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:48.
[0277] Optionally, the antigen-binding site which binds to CEA
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:43, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:45; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:46, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:47, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:48. In some embodiments, CDR-H1 may have the
sequence GFTFTDYYMN (SEQ ID NO.: 151).
[0278] In another aspect, the antigen-binding site which binds to
CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:43; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:44; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:45; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:46; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:47; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:48. In some embodiments, CDR-H1 may have the sequence GFTFTDYYMN
(SEQ ID NO.: 151).
[0279] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-CEA antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0280] In another embodiment, the antigen-binding site which binds
to CEA comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:49. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to CEA, preferably with the affinity as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:49. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the antigen-binding site
which binds to CEA comprises the VH sequence in SEQ ID NO:49,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:43 or the sequence GFTFTDYYMN (SEQ ID NO.: 151), (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:44, and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:45.
[0281] In another embodiment, the antigen-binding site which binds
to CEA comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:50. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but the antigen-binding site comprising
that sequence retains the ability to bind to CEA, preferably with
the affinity set out above. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:50. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the antigen-binding site for CEA comprises
the VL sequence in SEQ ID NO:50, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three CDRs selected from (a) CDR-L1
comprising the amino acid sequence of SEQ ID NO:46; (b) CDR-L2
comprising the amino acid sequence of SEQ ID NO:47; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:48.
[0282] In another embodiment, the antigen-binding site which binds
to CEA comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:49 and SEQ ID NO:50, respectively, including
post-translational modifications of those sequences.
iv) In a still further particular embodiment, the antigen-binding
site which binds to CEA may comprise at least one, two, three,
four, five, or six CDRs selected from (a) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:59; (b) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:60; (c) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:61; (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:62; (e) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:63; and (f) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:64.
[0283] Optionally, the antigen-binding site which binds to CEA may
comprise at least one, at least two, or all three VH CDR sequences
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:60; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:61.
[0284] Optionally, the antigen-binding site which binds to CEA
comprises at least one, at least two, or all three VL CDRs
sequences selected from (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:62; (b) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:63; and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:64.
[0285] Optionally, the antigen-binding site which binds to CEA
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:59, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:60, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:61; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:62, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:63, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:64.
[0286] In another aspect, the antigen-binding site which binds to
CEA comprises (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:59; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:60; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:61; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:62; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:63; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:64.
[0287] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-CEA antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0288] In another embodiment, the antigen-binding site which binds
to CEA comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:65. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to CEA, preferably with the affinity as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:65. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the antigen-binding site
which binds to CEA comprises the VH sequence in SEQ ID NO:65,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:59, (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:60, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:61.
[0289] In another embodiment, the antigen-binding site which binds
to CEA comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:66. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but the antigen-binding site comprising
that sequence retains the ability to bind to CEA, preferably with
the affinity set out above. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:66. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the antigen-binding site for CEA comprises
the VL sequence in SEQ ID NO:66, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three CDRs selected from (a) CDR-L1
comprising the amino acid sequence of SEQ ID NO:62; (b) CDR-L2
comprising the amino acid sequence of SEQ ID NO:63; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:64.
[0290] In another embodiment, the antigen-binding site which binds
to CEA comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:65 and SEQ ID NO:66, respectively, including
post-translational modifications of those sequences.
v). In a still further particular embodiment, the antigen-binding
site which binds to CEA may comprise at least one, two, three,
four, five, or six CDRs selected from (a) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:156; (b) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:157 or 158; (c) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:159; (d) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:160, 161 or 162; (e) CDR-L2
comprising the amino acid sequence of SEQ ID NO:163, 164 or 165;
and (0 CDR-L3 comprising the amino acid sequence of SEQ ID
NO:166.
[0291] Optionally, the antigen-binding site which binds to CEA may
comprise:
[0292] VH CDR sequences (a) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:156; (b) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:157 or 158; and (c) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:159; and/or
[0293] VL CDRs sequences (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:160, 161 or 162; (b) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:163, 164 or 165; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:166.
[0294] In one embodiment, the antigen binding site for CEA
comprises a heavy chain variable region (VH) comprise the amino
acid sequence of SEQ ID NO: 167, or (more preferably) selected from
SEQ ID NO: 169, 170, 171, 172, 173 or 174, and a light chain
variable region (VL) comprising the amino acid sequence of SEQ ID
NO: 168 or (more preferably) selected from SEQ ID NO: 175, 176,
177, 178, 179 or 180.
[0295] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-CEA antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0296] In a particular aspect, the antigen binding domain capable
of binding to CEA comprises:
[0297] (a) a VH domain comprising an amino acid sequence of SEQ ID
NO:169 and a VL domain comprising an amino acid sequence of SEQ ID
NO:179, or
[0298] (b) a VH domain comprising an amino acid sequence of SEQ ID
NO:173 and a VL domain comprising an amino acid sequence of SEQ ID
NO:179, or
[0299] (c) a VH domain comprising an amino acid sequence of SEQ ID
NO:170 and a VL domain comprising an amino acid sequence of SEQ ID
NO:179, or
[0300] (d) a VH domain comprising an amino acid sequence of SEQ ID
NO:174 and a VL domain comprising an amino acid sequence of SEQ ID
NO:178, or
[0301] (e) a VH domain comprising an amino acid sequence of SEQ ID
NO:173 and a VL domain comprising an amino acid sequence of SEQ ID
NO:178, or
[0302] (f) a VH domain comprising an amino acid sequence of SEQ ID
NO:171 and a VL domain comprising an amino acid sequence of SEQ ID
NO:178, or
[0303] (g) a VH domain comprising an amino acid sequence of SEQ ID
NO:169 and a VL domain comprising an amino acid sequence of SEQ ID
NO:178.
[0304] In another embodiment, the antigen-binding site which binds
to CEA comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence as mentioned in a) to
g) above. In certain embodiments, a VH sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but
the antigen binding site comprising that sequence retains the
ability to bind to CEA, preferably with the affinity as set out
above. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted and/or deleted. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs).
[0305] In another embodiment, the antigen-binding site which binds
to CEA comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence as mentioned in a) to g) above.
In certain embodiments, a VL sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the
antigen-binding site comprising that sequence retains the ability
to bind to CEA, preferably with the affinity set out above. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted. In certain embodiments, the
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs).
[0306] In another embodiment, the antigen-binding site which binds
to CEA comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above.
F. Exemplary Antigen Binding Sites for Other Targets
[0307] In another particular embodiment of the present invention,
which may be combined with the embodiments discussed above (e.g.,
the binding sites for DOTA or DOTAM), the target antigen bound by
the first and second antibody may be GPRC5D or FAP.
[0308] Optionally, the antigen-binding site which binds to GPRC5D
or FAP may bind with a Kd value of 1 nM or less, 500 pM or less,
200 pM or less, or 100 pM or less for monovalent binding.
[0309] Exemplary GPRC5D-binding sequences are described below.
[0310] In one embodiment, the antigen-binding site which binds to
GPRC5D may comprise at least one, two, three, four, five, or six
CDRs selected from (a) CDR-H1 comprising the amino acid sequence of
SEQ ID NO:67; (b) CDR-H2 comprising the amino acid sequence of SEQ
ID NO:68; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:69; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:70; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:71; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:72.
[0311] Optionally, the antigen-binding site which binds to GPRC5D
may comprise at least one, at least two, or all three VH CDR
sequences selected from (a) CDR-H1 comprising the amino acid
sequence of SEQ ID NO:67; (b) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:68; and (c) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:69. Optionally, the antigen-binding site
which binds to GPRC5D comprises at least one, at least two, or all
three VL CDRs sequences selected from (a) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:70; (b) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:71; and (c) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:72.
[0312] Optionally, the antigen-binding site which binds to GPRC5D
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:67, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:68, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:69; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:70, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:71, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:72.
[0313] In another aspect, the antigen-binding site which binds to
GPRC5D comprises (a) CDR-H1 comprising the amino acid sequence of
SEQ ID NO:67; (b) CDR-H2 comprising the amino acid sequence of SEQ
ID NO:68; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:69; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:70; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:71; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:72.
[0314] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-GPRC5D antigen
binding site comprises CDRs as in any of the above embodiments, and
further comprises an acceptor human framework, e.g. a human
immunoglobulin framework or a human consensus framework.
[0315] In another embodiment, the antigen-binding site which binds
to GPRC5D comprises a heavy chain variable domain (VH) sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:73. In certain embodiments, a VH sequence having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to GPRC5D, preferably with the affinity as set out above. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO:73. In certain
embodiments, substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs). Optionally, the
antigen-binding site which binds to GPRC5D comprises the VH
sequence in SEQ ID NO:73, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three CDRs selected from: (a) CDR-H1
comprising the amino acid sequence of SEQ ID NO:67, (b) CDR-H2
comprising the amino acid sequence of SEQ ID NO:68, and (c) CDR-H3
comprising the amino acid sequence of SEQ ID NO:69.
[0316] In another embodiment, the antigen-binding site which binds
to GPRC5D comprises a light chain variable domain (VL) having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:74. In
certain embodiments, a VL sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the
antigen-binding site comprising that sequence retains the ability
to bind to GPRC5D, preferably with the affinity set out above. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO:74. In certain
embodiments, the substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs). Optionally, the
antigen-binding site for GPRC5D comprises the VL sequence in SEQ ID
NO:74, including post-translational modifications of that sequence.
In a particular embodiment, the VL comprises one, two or three CDRs
selected from (a) CDR-L1 comprising the amino acid sequence of SEQ
ID NO:70; (b) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:71; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:72.
[0317] In another embodiment, the antigen-binding site which binds
to GPRC5D comprises a VH as in any of the embodiments provided
above, and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:73 and SEQ ID NO:74, respectively, including
post-translational modifications of those sequences.
[0318] Exemplary FAP-binding sequences are described below.
[0319] In one embodiment, the antigen-binding site which binds to
FAP may comprise at least one, two, three, four, five, or six CDRs
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:75; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:76; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:77; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:78; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:79; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:80.
[0320] Optionally, the antigen-binding site which binds to FAP may
comprise at least one, at least two, or all three VH CDR sequences
selected from (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:75; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:76; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:77.
[0321] Optionally, the antigen-binding site which binds to FAP
comprises at least one, at least two, or all three VL CDRs
sequences selected from (a) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:78; (b) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:79; and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:80.
[0322] Optionally, the antigen-binding site which binds to FAP
comprises (a) a VH domain comprising at least one, at least two, or
all three VH CDR sequences selected from (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:75, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:76, and (iii) CDR-H3 comprising an
amino acid sequence selected from SEQ ID NO:77; and (b) a VL domain
comprising at least one, at least two, or all three VL CDR
sequences selected from (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:78, (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:79, and (c) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:80.
[0323] In another aspect, the antigen-binding site which binds to
FAP comprises (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:75; (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:76; (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:77; (d) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:78; (e) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:79; and (f) CDR-L3 comprising an amino acid sequence SEQ ID
NO:80.
[0324] In any of the above embodiments, the multispecific antibody
may be humanized. In one embodiment, the anti-FAP antigen binding
site comprises CDRs as in any of the above embodiments, and further
comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a human consensus framework.
[0325] In another embodiment, the antigen-binding site which binds
to FAP comprises a heavy chain variable domain (VH) sequence having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:81. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but the antigen
binding site comprising that sequence retains the ability to bind
to FAP, preferably with the affinity as set out above. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:81. In certain embodiments,
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the antigen-binding site
which binds to FAP comprises the VH sequence in SEQ ID NO:81,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three CDRs
selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ
ID NO:75, (b) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:76, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:77.
[0326] In another embodiment, the antigen-binding site which binds
to FAP comprises a light chain variable domain (VL) having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:82. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but the antigen-binding site comprising
that sequence retains the ability to bind to FAP, preferably with
the affinity set out above. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:82. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the antigen-binding site for FAP comprises
the VL sequence in SEQ ID NO:82, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three CDRs selected from (a) CDR-L1
comprising the amino acid sequence of SEQ ID NO:78; (b) CDR-L2
comprising the amino acid sequence of SEQ ID NO:79; and (c) CDR-L3
comprising the amino acid sequence of SEQ ID NO:80.
[0327] In another embodiment, the antigen-binding site which binds
to FAP comprises a VH as in any of the embodiments provided above,
and a VL as in any of the embodiments provided above. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:81 and SEQ ID NO:82, respectively, including
post-translational modifications of those sequences.
G. Antibody Formats
[0328] As described above, the present invention relates to a set
of antibodies comprising:
[0329] i) a first antibody that binds to an antigen expressed on
the surface of a target cell, and which further comprises a VH
domain of an antigen binding site for a radiolabelled compound, but
which does not comprise a VL domain of an antigen binding site for
the radiolabelled compound; and
[0330] ii) a second antibody that binds to the antigen expressed on
the surface of the target cell, and which further comprises a VL
domain of an antigen binding site for the radiolabelled compound,
but which does not comprise a VH domain of the antigen binding site
for the radiolabelled compound,
[0331] wherein said VH domain of the first antibody and said VL
domain of the second antibody are together capable of forming a
functional antigen binding site for the radiolabelled compound.
[0332] In some embodiments, the first and second antibody may each
comprise an Fc domain. The presence of an Fc region has benefits in
the context of radioimmunotherapy and radioimaging, e.g. prolonging
the protein's circulating half-life and/or resulting in higher
tumour uptake than may be observed with smaller fragments.
[0333] In some embodiments, where the Fc region is present, it may
be preferred that the Fc region is engineered to reduce or
eliminate effector function. This may include substitution of one
or more of Fc region residues 234, 235, 238, 265, 269, 270, 297,
327 and/or 329, e.g., one or more of 234, 235 and/or 329. In some
embodiments, the Fc region may be engineered to include the
substitution of Pro 329 to Gly, Leu 234 to Ala and/or Leu 235 to
Ala (numbering according to EU index).
[0334] In some embodiments, as discussed above, where the VH domain
of an antigen binding site for a radiolabelled compound is free at
its C-terminus (e.g., is not fused to another domain via its
C-terminus), then it may be extended by one or more residues to
avoid binding of HAVH autoantibodies. For instance, the extension
may be by 1-10 residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
residues. In one embodiment, it may be extended by one or more
alanine residues, optionally by one alanine residue. The VH
sequence may also be extended by an N-terminal portion of the CH1
domain, e.g., by 1-10 residues from the N-terminus of the CH1
domain, e.g., from the human IgG1 CH1 domain. (The first ten
residues of the human IgG1 CH1 domain are ASTKGPSVFP (SEQ ID NO.:
149), and so in one embodiment, from 1-10 residues may be taken
from the N-terminus of this sequence). For instance, in one
embodiment, the peptide sequence AST (corresponding to the first 3
residues of the IgG1 CH1 domain) is added to the C-terminus of the
VH region. In some embodiments, the first and/or the second
antibody may each be multivalent, e.g., bivalent for the target
antigen (e.g., the tumour associated antigen). This has the
advantage of increasing avidity.
[0335] In some embodiments, it may be preferred that when the first
and second antibody are associated, they form an antibody complex
which is monovalent for the radiolabelled compound. Thus, the first
antibody may comprise only one VH domain of an antigen binding site
for the radiolabelled compound, and the second antibody may
comprise only one VL domain of an antigen binding site for a
radiolabelled compound, so that together they form only one
complete functional binding site for the radiolabelled
compound.
[0336] The antibodies may each comprise i) at least one antibody
fragment comprising an antigen binding site specific for the target
antigen, ii) either a VL domain or a VH domain of the antigen
binding site for the radiolabelled compound, and iii) optionally a
Fc region. The antibody fragment may be for example at least one
Fv, scFv, Fab or cross-Fab fragment, comprising an antigen binding
site specific for the target antigen. The antibody fragment may be
fused to a) either a VL domain or a VH domain of the antigen
binding site for the radiolabelled compound or b) if the antibodies
comprise a Fc region, to a Fc region which is fused to either a VL
domain or a VH domain of the antigen binding site for the
radiolabelled compound. In some embodiments, the C-terminus of the
Fc region is fused to the N-terminus of the VL domain or VH
domain.
[0337] The fusion may be direct or indirect. In some embodiments,
the fusion may be via a linker. For instance, the Fc region may be
fused to the antibody fragment via the hinge region or another
suitable linker. Similarly, the connection of the VL or VH domain
of the antigen binding site for the radiolabelled compound to the
rest of the antibody structure may be made via a linker. The linker
may be a peptide of at least 5 amino acids, preferably 5 to 100,
more preferably 10 to 50 or 25 to 50 amino acids. The linker may be
a rigid linker or a flexible linker. In some embodiments, it is a
flexible linker comprising or consisting of Thr, Ser, Gly and/or
Ala residues. For example, it may comprise or consist of Gly and
Ser residues. In some embodiments it may have a repeating motif
such as (Gly-Gly-Gly-Gly-Ser)n, where n is for instance 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10. In another embodiment said peptide linker is
(GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or
6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3),
e.g., x=4 and n=2 or 3, e.g., with x=4, n=2. In some embodiments,
the linker may be or may comprise the sequence GGGGSGGGGSGGGGSGGGGS
(SEQ ID NO.: 31). Other linkers may be used and could be identified
by the skilled person.
[0338] In one particular embodiment, the first antibody may
comprise or consist of:
[0339] a) an scFv fragment, wherein the scFv fragment binds the
target antigen; and
[0340] b) a polypeptide comprising or consisting of [0341] i) an
antibody heavy chain variable domain (VH); or [0342] ii) an
antibody heavy chain variable domain (VH) and an antibody heavy
chain constant domain, wherein the C-terminus of the VH domain is
fused to the N terminus of the constant domain;
[0343] wherein said polypeptide is fused by the N-terminus of the
VH domain, preferably via a peptide linker, to the C-terminus of
scFv fragment.
[0344] The second antibody may comprise or consist of:
[0345] c) a second scFv binding the target antigen; and
[0346] d) a polypeptide comprising or consisting of [0347] i) an
antibody light chain variable domain (VL); or [0348] ii) an
antibody light chain variable domain (VL) and an antibody light
chain constant domain, wherein the C-terminus of the VL domain is
fused to the N-terminus of the constant domain; wherein said
polypeptide is fused by the N-terminus of the VL domain, preferably
via a peptide linker, to the C-terminus of scFv fragment.
[0349] The antibody heavy chain variable domain (VH) of the first
antibody and the antibody light chain variable domain (VL) of the
second antibody together form a functional antigen-binding site for
the radiolabelled compound, upon association of the two
antibodies.
[0350] Optionally, the polypeptide of part b (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0351] The target antigen-recognizing variable domains of the heavy
and light chain of an scFv can be connected by a peptide tether.
Such a peptide tether may comprise 1 to 25 amino acids, preferably
12 to 20 amino acids, preferably 12 to 16 or 15 to 20 amino acids.
The above described tether may comprise one or more (G3S) and/or
(G4S) motifs, in particular 1, 2, 3, 4, 5 or 6 (G3S) and/or (G4S)
motives, preferably 3 or 4 (G3S) and/or (G4S) motives, more
preferably 3 or 4 (G4S) motives.
[0352] Optionally, the first antibody may consist essentially of or
consist of the components (a) and (b) listed above and the second
antibody may consist or consist essentially of the components (c)
and (d) listed above. In any event, the first antibody does not
comprise an antibody light chain variable domain (VL) capable of
forming a functional antigen-binding site for the radiolabelled
compound in association with component (b) of the first antibody;
and the second antibody does not comprise an antibody heavy chain
variable (VH) domain capable of forming a functional
antigen-binding site for the radiolabelled compound in association
with component (d) of the second antibody.
[0353] In another particular embodiment, the first antibody may
comprise or consist of:
[0354] a) a Fab fragment binding the target antigen, and
[0355] b) a polypeptide comprising or consisting of [0356] i) an
antibody heavy chain variable domain (VH) of an antigen binding
site for a radiolabelled compound, or [0357] ii) an antibody heavy
chain variable domain (VH) of an antigen binding site for a
radiolabelled compound and an antibody heavy chain constant domain,
wherein the C-terminus of VH domain is fused to the N-terminus of
the constant domain; [0358] wherein the polypeptide is fused by the
N-terminus of the VH domain, preferably via a peptide linker, to
the C terminus of the CL or CH1 domain of the Fab fragment. The
second antibody may comprise or consist of:
[0359] c) a Fab fragment binding the target antigen, and
[0360] d) a polypeptide comprising or consisting of [0361] iii) an
antibody light chain variable domain (VL) of an antigen binding
site for a radiolabelled compound, or [0362] iv) an antibody light
chain variable domain (VL) of an antigen binding site for a
radiolabelled compound and an antibody light chain constant domain,
wherein the C-terminus of the VL domain is fused to the N-terminus
of the constant domain;
[0363] wherein the polypeptide is fused by the N-terminus of the VL
domain, preferably via a peptide linker, to the C-terminus of the
CL or CH1 domain of the Fab fragment.
[0364] The antibody heavy chain variable domain (VH) of the
polypeptide of (b) and antibody light chain variable domain (VL) of
polypeptide of (d) together form a functional antigen-binding site
for the radiolabelled compound (i.e., upon association of the two
antibodies).
[0365] Optionally, the polypeptide of part b (i) may additionally
comprise one or more residues at the C-terminus of the VH domain as
described above, optionally, one or more alanine residues,
optionally a single alanine residue. Optionally, the additional
residues may be an N-terminal portion of the CH1 domain as
described above, e.g., 1-10 residues from the N-terminus of the CH1
domain, e.g., from the human IgG1 CH1 domain. For instance, the
additional residues may be AST.
[0366] Optionally, the first antibody may consist essentially of or
consist of the components (a) and (b) listed above and the second
antibody may consist or consist essentially of the components (c)
and (d) listed above. In any event, the first antibody does not
comprise an antibody light chain variable domain (VL) capable of
forming a functional antigen-binding site for the radiolabelled
compound in association with component (b) of the first antibody;
and the second antibody does not comprise an antibody heavy chain
variable (VH) domain capable of forming a functional
antigen-binding site for the radiolabelled compound in association
with component (d) of the second antibody.
[0367] The chain of the Fab fragment which is fused to the
polypeptide can be independently selected for the first and for the
second anybody. Thus, in one embodiment, the polypeptide of (b) is
fused to the C-terminus of the CH1 domain of the Fab fragment of
the first antibody, and the polypeptide of (d) is fused to the
C-terminus of the CH1 domain of the Fab fragment of the second
antibody. In another embodiment, the polypeptide of (b) is fused to
the C-terminus of the CL domain of the Fab fragment of the first
antibody, and the polypeptide of (d) is fused to the C-terminus of
the CL domain of the Fab fragment of the second antibody. In
another embodiment, the polypeptide of (b) is fused to the
C-terminus of the CH1 domain of the Fab fragment of the first
antibody, and polypeptide of (d) is fused to the C-terminus of the
CL domain of the Fab fragment of the second antibody. In a further
embodiment, polypeptide of (b) is fused to the C-terminus of the CL
domain of the Fab fragment of the first antibody, and the
polypeptide of (d) is fused to the C-terminus of the CH1 domain of
the Fab fragment of the second antibody.
[0368] As noted above, in some embodiments, the first and/or the
second antibody may each be multivalent, e.g., bivalent for the
target antigen (e.g., the tumour associated antigen). This has the
advantage of increasing avidity. The antibodies may be multivalent,
e.g., bivalent, and may each be monospecific for a particular
epitope (which may be the same epitope for the first and second
antibody, or may be different for the first and second antibody).
Thus, in some embodiments, the first antibody may comprise i) two
or more antibody fragments comprising an antigen binding site
specific for the same epitope of the target antigen, ii) either a
VL domain or a VH domain of the antigen binding site for the
radiolabelled compound (but not both), and iii) optionally a Fc
region. The second antibody may comprise i) two or more antibody
fragments comprising an antigen binding site specific for the same
epitope of the target antigen, ii) either a VL domain or a VH
domain of the antigen binding site for the radiolabelled compound
(but not both), and iii) optionally a Fc region. As stated above,
the epitope may be the same for the first and second antibody, or
may be different for the first and second antibody
[0369] For example, each of the first and the second antibody may
comprise a tandem Fab, i.e., two Fab fragments, which are connected
via a peptide tether (Fab-tether-Fab), wherein the first Fab is
connected via its C-terminus to the N-terminus of the second
Fab.
[0370] In one embodiment, the first antibody comprises
[0371] a) a tandem Fab comprising two Fab fragments, wherein the
first and the second Fab fragment bind the same target antigen
("target antigen A") and the epitope bound by the first Fab
fragment is the same as the epitope bound by the second Fab
fragment, and wherein the first and the second Fab fragment are
connected via a peptide tether, wherein the first Fab is connected
via its C-terminus to the N-terminus of the second Fab; and
[0372] b) a polypeptide comprising or consisting of [0373] i) an
antibody heavy chain variable domain (VH); or [0374] ii) an
antibody heavy chain variable domain (VH) and an antibody constant
domain (CH1), wherein the C-terminus of VH domain is fused to the
N-terminus of the CH1 domain;
[0375] wherein said polypeptide is fused by the N-terminus of the
VH domain, preferably via a peptide linker, to the C-terminus of
the CL or CH1 domain of the second Fab fragment;
[0376] and the second antibody comprises
[0377] c) a tandem Fab comprising two Fab fragments, wherein the
first and the second Fab fragment bind target antigen A and the
epitope bound by the first Fab fragment is the same as the epitope
bound by the second Fab fragment, and wherein the first and the
second Fab fragment are connected via a peptide tether, wherein the
first Fab is connected via its C-terminus to the N-terminus of the
second Fab; and
[0378] d) a polypeptide comprising or consisting of
[0379] i) an antibody light chain variable domain (VL); or
[0380] ii) an antibody light chain variable domain (VL) and an
antibody light chain constant domain (CL), wherein the C-terminus
of VH domain is fused to the N-terminus of the constant domain;
[0381] wherein said polypeptide is fused by the N-terminus of the
VL domain, preferably via a peptide linker, to the C-terminus of
the CL or CH1 domain of the second Fab fragment.
[0382] The antibody heavy chain variable domain (VH) of part b (in
the first antibody) and the antibody light chain variable domain
(VL) of part (d) (in the second antibody) together form a
functional antigen-binding site for the radiolabelled compound,
i.e., upon association of the two antibodies.
[0383] Optionally, the polypeptide of part b (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0384] The chain of the Fab tandem which is fused to the
polypeptide (i.e., whether the polypeptide is fused to the CL or
the CH1 domain of the second Fab fragment) can be independently
selected for the first and for the second anybody.
[0385] As described above, the first Fab fragment of the Fab tandem
is connected to the N-terminus of the second Fab fragment. In one
embodiment, the C-terminus of the heavy chain fragment of the first
Fab fragment is connected to the N-terminus of the heavy-chain
fragment or light chain fragment of the second Fab fragment. In
another embodiment, the C-terminus light chain fragment of the
first Fab fragment is connected to the N-terminus of the
heavy-chain fragment or light chain fragment of the second Fab
fragment. Thus, in some embodiments the Fab tandem of the first
and/or second antibody may comprise three chains as follows:
[0386] 1) the light chain fragment ((VLCL)1) of the first Fab
fragment, the heavy chain fragment of the first Fab fragment
connected to the heavy chain fragment of the second Fab fragment
via a peptide tether ((VHCH1)1-tether-(VHCH1)2) and the light chain
fragment of the second Fab fragment ((VLCL)2); or
[0387] 2) the light chain fragment of the first Fab fragment
((VLCL)1), the heavy chain fragment of the first Fab fragment
connected to the light chain fragment of the second Fab fragment
via a peptide tether ((VHCH1)1-tether-(VLCL)2) and the heavy chain
fragment of the second Fab fragment ((VH-CH1)2); or
[0388] 3) the heavy chain fragment (VHCH1) of the first Fab
fragment, the light chain fragment of the first Fab fragment
connected to the light chain fragment of the second Fab fragment
via a peptide tether ((VLCL)1-tether-(VLCL)2) and the heavy chain
fragment of the second Fab fragment; or
[0389] 4) the heavy chain fragment (VHCH1) of the first Fab
fragment, the light chain fragment of the first Fab fragment
connected to the heavy chain fragment of the second Fab fragment
via a peptide tether ((VLCL)1-tether-(VHCH1)2) and the light chain
fragment of the second Fab fragment ((VLCL)2).
[0390] In another embodiment, the first and/or second antibody may
each bind more than one, optionally two, different epitopes of the
target antigen. Thus, one or both of the antibodies may be
biparatopic for the target antigen. In some embodiments, the first
and second antibody may each comprise i) an antibody fragment
comprising an antigen binding site specific for a first epitope of
target antigen A; ii) an antibody fragment comprising an antigen
binding site for a second epitope of target antigen A, iii) either
a VL domain or a VH domain of the antigen binding site for the
radiolabelled compound (but not both), and iv) optionally a Fc
region.
[0391] In such embodiments, correct assembly of the light chains
with their respective heavy chain can be assisted by using
cross-mab technology. For instance, in one embodiment, each
antibody may comprise a tandem Fab comprising one Fab and one
cross-Fab, in which one fragment selected from the Fab and the
cross-Fab is specific for a first epitope, and the other is
specific for a second epitope.
[0392] In one particular example, the first antibody may
comprise:
[0393] a) a tandem Fab comprising a first fragment and a second
fragment, wherein the first fragment is connected by its C-terminus
via a peptide tether to the N-terminus of the second fragment,
wherein the first fragment binds a first epitope of target antigen
A and the second fragment binds a second epitope of target antigen
A, and wherein one of the fragments selected from the first and
second fragments is a Fab and the other is a cross-Fab,
[0394] b) a polypeptide comprising or consisting of [0395] i) an
antibody heavy chain variable domain (VH); or [0396] ii) an
antibody heavy chain variable domain (VH) and an antibody heavy
chain constant domain (CH1), wherein the C-terminus of VH domain is
fused to the N-terminus of the CH1 domain;
[0397] wherein said polypeptide is fused by the N-terminus of the
VH domain, preferably via a peptide linker, to the C-terminus of
one of the chains of the second fragment.
[0398] The second antibody may comprise
[0399] c) a tandem Fab comprising a first fragment and a second
fragment, wherein the first fragment is connected by its C-terminus
to the N-terminus of the second fragment, wherein the first
fragment binds a first epitope of target antigen A and the second
fragment binds a second epitope of target antigen A, and wherein
one of the fragments selected from the first and second fragments
is a Fab and the other is a cross-Fab; and
[0400] d) a polypeptide comprising or consisting of
[0401] i) an antibody light chain variable domain (VL); or
[0402] ii) an antibody light chain variable domain (VL) and an
antibody light chain constant domain (CL), wherein the C-terminus
of VL domain is fused to the N-terminus of the light chain constant
domain
[0403] wherein said polypeptide is fused by the N-terminus of the
VL domain, preferably via a peptide linker, to the C-terminus of
one of the chains of the second fragment.
[0404] The antibody heavy chain variable domain (VH) of the first
antibody and the antibody light chain variable domain (VL) of the
second antibody together form a functional antigen-binding site for
the radiolabelled compound.
[0405] Optionally, the polypeptide of part b (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0406] Either the first or second fragment can be the cross-Fab, as
long as the tandem Fab comprises one conventional Fab and one cross
Fab.
[0407] In any of the tandem Fab embodiments described above
(including those involving cross-Fabs), optionally, the first
antibody may consist essentially of or consist of the components
(a) and (b) and the second antibody may consist or consist
essentially of the components (c) and (d). In any event, the first
antibody does not comprise an antibody light chain variable domain
(VL) capable of forming a functional antigen-binding site for the
radiolabelled compound in association with component (b) of the
first antibody; and the second antibody does not comprise an
antibody heavy chain variable (VH) domain capable of forming a
functional antigen-binding site for the radiolabelled compound in
association with component (d) of the second antibody.
[0408] In any of the tandem Fab embodiments (including those
involving cross-Fabs), the peptide tether connecting the Fab
fragments in the first and second antibody may be a peptide with an
amino acid sequence with a length of at least 5 amino acids,
preferably with a length of 5 to 100, more preferably of 10 to 50
amino acids. In one embodiment said peptide linker is (GxS)n or
(GxS)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and
m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3),
preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In one
embodiment said peptide tether is (G4S).sub.2.
[0409] As noted above, in some embodiments, the first and second
antibody may each comprise an Fc domain, optionally engineered to
reduce or eliminate effector function.
[0410] In one embodiment, each of the first and second antibody may
comprise i) an Fc domain, ii) at least one antibody fragment, such
as an scFv, Fv, Fab or cross-Fab fragment, comprising an antigen
binding site specific for the target antigen and iii) either a VL
domain or a VH domain of the antigen binding site for the
radiolabelled compound (but not both).
[0411] Optionally, the antibodies comprising the Fc domain may be
monovalent in respect of binding to the target antigen. In other
embodiments, they may be multivalent, e.g., bivalent. The first and
second antibodies may each be multivalent and monospecific for the
same epitope of the target antigen. In still other embodiments, the
first and second antibodies may each have binding sites for
different epitopes of the target antigen--e.g., they may be
biparatopic.
[0412] The antibody fragment may be an scFv. Thus, in one
embodiment, the first antibody may comprise or consist of:
[0413] a) an scFv fragment, wherein the scFv fragment binds the
target antigen;
[0414] b) an Fc domain; and
[0415] c) a polypeptide comprising or consisting of
[0416] i) an antibody heavy chain variable domain (VH); or
[0417] ii) an antibody heavy chain variable domain (VH) and an
antibody heavy chain constant domain (CH1), wherein the C-terminus
of the VH domain is fused to the N-terminus of the constant
domain;
[0418] wherein the scFv of (a) is fused to the N-terminus of the Fc
domain, and wherein the polypeptide of c) is fused by the
N-terminus of the VH domain to the C-terminus of the Fc domain,
preferably via a peptide linker.
[0419] Optionally, the polypeptide of part c (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG.sub.1 CH1 domain. For instance, the additional residues
may be AST.
[0420] The second antibody may comprise or consist of:
[0421] d) a second scFv binding the target antigen;
[0422] e) an Fc domain; and
[0423] f) a polypeptide comprising or consisting of
[0424] i) an antibody light chain variable domain (VL); or
[0425] ii) an antibody light chain variable domain (VL) and an
antibody light chain constant domain (CL), wherein the C-terminus
of the VL domain is fused to the N-terminus of the constant
domain;
[0426] wherein the scFv of (d) is fused to the N-terminus of the Fc
domain, and wherein the polypeptide of (0 is fused by the
N-terminus of the VH domain to the C-terminus of the Fc domain,
preferably via a peptide linker.
[0427] In another embodiment, the first and second antibody may
each be a one-armed IgG comprising a Fab for the target antigen
(e.g., a single Fab for the target antigen) and an Fc domain. Thus,
the first antibody may comprise or consist of:
[0428] i) a complete light chain fragment;
[0429] ii) a complete heavy chain;
[0430] iii) an additional Fc chain lacking Fd; and
[0431] iv) a polypeptide comprising or consisting of the VH domain
of the antigen binding site for the radiolabeled compound;
wherein the light chain of (i) and the heavy chain of (ii) together
provide an antigen binding site for the target antigen; and wherein
the polypeptide comprising or consisting of the VH domain of the
antigen binding site for the radiolabeled compound is fused by its
N-terminus, preferably via a linker, to the C-terminus of either
(ii) or (iii).
[0432] The second antibody may comprise or consist of
[0433] v) a complete light chain fragment;
[0434] vi) a complete heavy chain;
[0435] vii) an additional Fc chain lacking Fd; and
[0436] viii) a polypeptide comprising or consisting of the VL
domain of the antigen binding site for the radiolabeled
compound;
[0437] wherein the light chain of (v) and the heavy chain of (vi)
together provide an antigen binding site for the target antigen;
and wherein the polypeptide comprising or consisting of the VL
domain of the antigen binding site for the radiolabeled compound is
fused by its N-terminus, preferably via a linker, to the C-terminus
of either (vi) or (vii).
[0438] The polypeptide comprising or consisting of the VH domain of
the antigen binding site for the radiolabeled compound may be a
polypeptide comprising or consisting of
[0439] i) an antibody heavy chain variable domain (VH), in which
case the polypeptide may additionally comprise one or more residues
at the C-terminus of the VH domain, optionally, one or more alanine
residues, optionally a single alanine residue, or optionally an
N-terminal portion of the CH1 domain as described above; or
[0440] ii) an antibody heavy chain variable domain (VH) and an
antibody heavy chain constant domain (CH1), wherein the C-terminus
of VH domain is fused to the N-terminus of the CH1 domain.
[0441] The polypeptide comprising or consisting of the VL domain of
the antigen binding site for the radiolabeled compound may be a
polypeptide comprising or consisting of [0442] i) an antibody heavy
chain variable domain (VL); or [0443] ii) an antibody heavy chain
variable domain (VL) and an antibody light chain constant domain,
wherein the C-terminus of VL domain is fused to the N-terminus of
the constant domain.
[0444] When the first and second antibodies are heterodimers, e.g.,
as for one-armed IgGs, their assembly may be assisted by the use of
knob-into-hole technology, as described further below.
[0445] In another embodiment, the antibodies may each comprise a
tandem Fab as described above (e.g., comprising two Fab fragments,
wherein the first and the second Fab fragment both bind the same
epitope of target antigen A; or comprising a Fab and a cross Fab
wherein one of them binds a first epitope of target antigen A and
the other binds a second epitope of target antigen A), wherein the
Fab tandem is fused (e.g., via its C-terminus) to the N-terminus of
an Fc domain, and wherein peptide comprising or consisting of the
VH or VL domain of the antigen binding site for the radiolabelled
compound is fused (e.g., via its N-terminus) to the C-terminus of
the Fc domain.
[0446] Thus, the first antibody may comprise or consist of:
[0447] a) a tandem Fab selected from [0448] i) a tandem Fab
comprising two Fab fragments, wherein the first and the second Fab
fragment bind target antigen A and the epitope bound by the first
Fab fragment is the same as the epitope bound by the second Fab
fragment, and wherein the first and the second Fab fragment are
connected via a peptide tether, wherein the first Fab is connected
via its C-terminus to the N-terminus of the second Fab; and [0449]
ii) a tandem Fab comprising a first fragment and a second fragment,
wherein the first fragment is connected by its C-terminus via a
peptide tether to the N-terminus of the second fragment, wherein
the first fragment binds a first epitope of target antigen A and
the second fragment binds a second epitope of target antigen A, and
wherein one of the fragments selected from the first and second
fragments is a Fab and the other is a cross-Fab;
[0450] b) an Fc domain; and
[0451] c) a polypeptide comprising or consisting of: [0452] i) an
antibody heavy chain variable domain (VH); or [0453] ii) an
antibody heavy chain variable domain (VH) and an antibody heavy
chain constant domain (CH1), wherein the C-terminus of VH domain is
fused to the N-terminus of the CH1 domain, wherein the tandem Fab
is fused to the N-terminus of one of the chains of the Fc domain,
and the polypeptide of c) is fused by the N-terminus of the VH
domain to the C-terminus of one of the chains of the Fc domain,
preferably via a peptide linker.
[0454] Optionally, the polypeptide of part c (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0455] The second antibody may comprise or consist of:
[0456] d) a tandem Fab selected from: [0457] i) a tandem Fab
comprising two Fab fragments, wherein the first and the second Fab
fragment bind target antigen A and the epitope bound by the first
Fab fragment is the same as the epitope bound by the second Fab
fragment, and wherein the first and the second Fab fragment are
connected via a peptide tether, wherein the first Fab is connected
via its C-terminus to the N-terminus of the second Fab; and [0458]
ii) a tandem Fab comprising a first fragment and a second fragment,
wherein the first fragment is connected by its C-terminus via a
peptide tether to the N-terminus of the second fragment, wherein
the first fragment binds a first epitope of target antigen A and
the second fragment binds a second epitope of target antigen A, and
wherein one of the fragments selected from the first and second
fragments is a Fab and the other is a cross-Fab;
[0459] e) an Fc domain; and
[0460] f) a polypeptide comprising or consisting of: [0461] i) an
antibody heavy chain variable domain (VL); or [0462] ii) an
antibody heavy chain variable domain (VL) and an antibody light
chain constant domain, wherein the C-terminus of VL domain is fused
to the N-terminus of the light chain constant domain,
[0463] wherein the tandem Fab of (d) is fused to the N-terminus one
of the chains of the Fc domain, and the polypeptide of (f) is fused
by the N-terminus of the VL domain to the C-terminus of one of the
chains of the Fc domain, preferably via a peptide linker.
[0464] The VH domain of the first antibody and the VL domain of the
second antibody together form an antigen binding site for the
radiolabelled compound, i.e., upon association of the two
antibodies.
[0465] If the first antibody comprises a tandem Fab according to
(a)(i), then it will generally be the case that the second antibody
will comprise a tandem Fab according to d (i); if the first
antibody comprises a tandem Fab according to (a)(ii), then it will
generally be the case that the second antibody will comprise a
tandem Fab according to d (ii).
[0466] The tandem Fab may be generally as described above. For
instance, the tether linking the two fragments of the tandem Fab
may be as described above. The tandem Fab may be composed of any of
the sets of chains set out above. Generally, the heavy chain
fragment of the second Fab (which may be a cross-Fab) can be linked
to the Fc domain.
[0467] In a further embodiment, each of the first and second
antibody may comprise a) an Fc domain b) at least one antibody
fragment, such as an scFv, Fv, Fab or cross-Fab fragment,
comprising an antigen binding site for the target antigen and c) a
polypeptide comprising either a VL domain or a VH domain of the
antigen binding site for the radiolabelled compound (but not both),
wherein the C-terminus of the antibody fragment of (b) is fused to
the N-terminus of one chain of the Fc domain, and the C-terminus of
the polypeptide of (c) is fused to the N-terminus of the other
chain of the Fc domain. The fusion of the antibody fragment of (b)
is preferably via the hinge region. The fusion of the polypeptide
of (c) may be via a linker positioned between the C-terminus of
polypeptide and the N-terminus of the Fc region and/or via some or
all of the upper hinge region (e.g., the Asp221 and residues
C-terminal thereto according to the EU numbering index). In one
embodiment, the antibody fragment of (b) may be a Fab fragment. In
one embodiment, in the first antibody, the polypeptide of (c)
consists of the VH domain of the antigen binding site for the
radiolabelled compound; and in the second antibody the polypeptide
of (c) consists of the VL domain of the antigen binding site for
the radiolabelled compound.
[0468] Thus, in one embodiment, the first antibody may comprise or
consist of:
[0469] i) a complete light chain;
[0470] ii) a complete heavy chain;
[0471] iii) an additional Fc chain; and
[0472] iv) a polypeptide comprising or consisting of the VH domain
of the antigen binding site for the radiolabeled compound;
[0473] wherein the light chain of (i) and the heavy chain of (ii)
together provide an antigen binding site for the target antigen;
and wherein the polypeptide comprising or consisting of the VH
domain of the antigen binding site for the radiolabeled compound is
fused by its C-terminus, preferably via a linker, to the N-terminus
of (iii).
[0474] The second antibody may comprise or consist of
[0475] v) a complete light chain;
[0476] vi) a complete heavy chain;
[0477] vii) an additional Fc chain; and
[0478] viii) a polypeptide comprising or consisting of the VL
domain of the antigen binding site for the radiolabeled
compound;
[0479] wherein the light chain of (v) and the heavy chain of (vi)
together provide an antigen binding site for the target antigen;
and wherein the polypeptide comprising or consisting of the VL
domain of the antigen binding site for the radiolabeled compound is
fused by its c-terminus, preferably via a linker, to the N-terminus
of (vii).
[0480] The linker may comprise any flexible linker as known to the
person skilled in the art, e.g., the linker GGGGSGGGGSGGGGSGGSGG
(SEQ ID NO.: 152). The linker may further include part of all of
the upper hinge region, e.g., may extend from Asp221 to the start
of the Fc chain (e.g., at Cys226).
[0481] In a still further embodiment, the first and/or second
antibody each comprise a full length antibody having an antigen
binding site for the target antigen, and further comprise either a
VL domain or a VH domain of the antigen binding site for the
radiolabelled compound.
[0482] In one particular embodiment, the first antibody may
comprise:
[0483] a) a first full length antibody consisting of two antibody
heavy chains and two antibody light chains, wherein at least one
arm of the full length antibody binds to target antigen A; and
[0484] b) a polypeptide comprising or consisting of [0485] i) an
further antibody heavy chain variable domain (VH); or [0486] ii) a
further antibody heavy chain variable domain (VH) and an further
antibody constant domain (CH1), wherein the C-terminus of VH domain
is fused to the N-terminus of the CH1 domain, [0487] wherein said
polypeptide is fused by the N-terminus of the VH domain, preferably
via a peptide linker, to the C-terminus of one of the two heavy
chains of said first full-length antibody.
[0488] The second antibody may comprise
[0489] c) a second full length antibody consisting of two antibody
heavy chains and two antibody light chains, wherein at least one
arm of the full length antibody binds to target antigen A; and
[0490] d) a polypeptide comprising or consisting of [0491] i) a
further antibody light chain variable domain (VL); or [0492] ii) a
further antibody light chain variable domain (VL) and a further
antibody light chain constant domain (CL), wherein the C-terminus
of VL domain is fused to the N-terminus of the CL domain, [0493]
wherein said polypeptide is fused by the N-terminus of the VL
domain, preferably via a peptide linker, to the C-terminus of one
of the two heavy chains of said second full-length antibody.
[0494] The antibody heavy chain variable domain (VH) of the first
antibody and the antibody light chain variable domain (VL) of the
second antibody together form a functional antigen-binding site for
the radiolabelled compound, i.e., upon association of the two
antibodies.
[0495] Optionally, the polypeptide of part b (i) may additionally
comprise one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. Optionally, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0496] Optionally, the first antibody may consist essentially of or
consist of the components (a) and (b) listed above, and the second
antibody may consist essentially of or consist of the components
(c) and (d) listed above. In any event, the first antibody does not
comprise an antibody light chain variable domain (VL) capable of
forming a functional antigen-binding site for the radiolabelled
compound in association with component (b) of the first antibody;
and the second antibody does not comprise an antibody heavy chain
variable (VH) domain capable of forming a functional
antigen-binding site for the radiolabelled compound in association
with component (b) of the second antibody.
[0497] It may be preferred that both arms of the full length
antibody have binding specificity for target antigen A. Where the
antibody is bivalent for the target antigen, both arms of the full
length antibody may bind to the same epitope of target antigen
A.
[0498] In another embodiment, the antibody may be biparatopic for
the target antigen; e.g., one arm of the full length antibody may
bind to a first epitope of target antigen A and one arm may bind to
a second epitope of target antigen A. In such embodiments, one arm
of the antibody may comprise a Fab and one arm may comprise a
cross-Fab, to assist in correct assembly of the light chains with
their respective heavy chain. Thus, in one embodiment, the first
heavy chain of the full length antibody may comprise a VL domain in
place of the VH domain (e.g., VL-CH1-hinge-CH2-CH3) and the first
light chain may comprise a VH domain exchanged for the VL domain
(e.g., VH-CL), or the first heavy chain may comprise a CL domain in
place of the HC1 domain (e.g., VH-CL-hinge-CH2-CH3) and the first
light chain may comprise a CH1 domain in place of the CL domain
(e.g., VL-CH1). In this embodiment, the second heavy chain and the
second light chain have the conventional domain structure (e.g.,
VH-CH1-hinge-CH2-CH3 and VL-CL, respectively). In an alternative
embodiment, the second heavy chain of the full length antibody may
comprise a VL domain in place of the VH domain (e.g.,
VL-CH1-hinge-CH2-CH3) and the second light chain may comprise a VH
domain exchanged for the VL domain (e.g., VH-CL), or the second
heavy chain may comprise a CL domain in place of the HC1 domain
(e.g., VH-CL-hinge-CH2-CH3) and the second light chain may comprise
a CH1 domain in place of the CL domain (e.g., VL-CH1). In this
embodiment, the first heavy chain and the first light chain have
the conventional domain structure.
[0499] In some embodiments, correct assembly of the light chains
with their respective heavy chain can additionally or alternatively
be assisted by using charge modification, as discussed further
below.
[0500] Correct assembly of heterodimeric heavy chains can be
assisted by knob-into-hole technology.
[0501] As used herein, the term "full length antibody" denotes an
antibody consisting of two "full length antibody heavy chains" and
two "full length antibody light chains". A "full length antibody
heavy chain" may be a polypeptide consisting in N-terminal to
C-terminal direction of an antibody heavy chain variable domain
(VH), an antibody constant heavy chain domain 1 (CH1), an antibody
hinge region (HR), an antibody heavy chain constant domain 2 (CH2),
and an antibody heavy chain constant domain 3 (CH3), abbreviated as
VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant
domain 4 (CH4) in case of an antibody of the subclass IgE.
Preferably the "full length antibody heavy chain" is a polypeptide
consisting in N-terminal to C-terminal direction of VH, CH1, HR,
CH2 and CH3. The possibility of cross-Mab formation is not intended
to be excluded by the reference to "full length"--thus, the heavy
chain may have the VH domain swapped for a VL domain, or the CH1
domain swapped for a CL domain. A "full length antibody light
chain" may be a polypeptide consisting in N-terminal to C-terminal
direction of an antibody light chain variable domain (VL), and an
antibody light chain constant domain (CL), abbreviated as VL-CL.
Alternatively, in the case of a cross-Mab, the VL domain may be
swapped for a VH domain or the CL domain may be swapped for a CH1
domain. The antibody light chain constant domain (CL) can be
.kappa. (kappa) or .gamma. (lambda). The two full length antibody
chains are linked together via inter-polypeptide disulfide bonds
between the CL domain and the CH1 domain and between the hinge
regions of the full length antibody heavy chains. Examples of
typical full length antibodies are natural antibodies like IgG
(e.g. IgG1 and IgG2), IgM, IgA, IgD, and IgE.) The full length
antibodies according to the invention can be from a single species
e.g. human, or they can be chimerized or humanized antibodies. The
full length antibodies described herein comprise two antigen
binding sites each formed by a pair of VH and VL, which may in some
embodiments both specifically bind to the same antigen, or may bind
to different antigens. The C-terminus of the heavy or light chain
of said full length antibody denotes the last amino acid at the
C-terminus of said heavy or light chain.
[0502] The N-terminus of the antibody heavy chain variable domain
(VH) of the polypeptide under b) and the antibody light chain
variable domain (VL) of the polypeptide under d) denotes the last
amino acid at the N-terminus of VH or VL domain.
[0503] Techniques which are known for making multispecific
antibodies can also be used to make any of the heterodimers
described herein. These include, but are not limited to,
recombinant co-expression of two immunoglobulin heavy chain-light
chain pairs having different specificities (see Milstein and
Cuello, Nature 305: 537 (1983)) and "knob-in-hole" engineering
(see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol.
Biol. 270:26 (1997)). Other methods include engineering
electrostatic steering effects for making antibody Fc-heterodimeric
molecule (see, e.g., WO 2009/089004); cross-linking two or more
antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and
Brennan et al., Science, 229: 81 (1985)); using leucine zippers
(see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)
and WO 2011/034605); and using the common light chain technology
for circumventing the light chain mis-pairing problem (see, e.g.,
WO 98/50431).
[0504] The CH3 domains of the full length antibody as described
above can be altered by the "knob-into-holes" technology which is
described in detail with several examples in e.g. WO 96/027011,
Ridgway, J. B., et al., Protein Eng 9 (1996) 617-621; and Merchant,
A. M., et al., Nat Biotechnol 16 (1998) 677-681. In this method the
interaction surfaces of the two CH3 domains are altered to increase
the heterodimerisation of both heavy chains containing these two
CH3 domains. Each of the two CH3 domains (of the two heavy chains)
can be the "knob", while the other is the "hole". For instance one
comprises called "knob mutations" (T366W and optionally one of
S354C or Y349C) and the other comprises the so-called "hole
mutations" (T366S, L368A and Y407V and optionally Y349C or 5354C)
(see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73)
according to EU index numbering.
[0505] The introduction of a disulfide bridge may additionally or
alternatively be used to stabilize the heterodimers (Merchant, A.
M., et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al.,
J. Mol. Biol. 270 (1997) 26-35) and increase the yield.
[0506] Thus in some embodiments the first and/or second antibody is
further characterized in that: the CH3 domain of one heavy chain of
the full length antibody and the CH3 domain of the other heavy
chain of the full length antibody each meet at an interface which
comprises an original interface between the antibody CH3 domains;
wherein said interface is altered to promote the formation of the
antibody, wherein the alteration is characterized in that:
[0507] a) the CH3 domain of one heavy chain is altered, so that
within the original interface the CH3 domain of one heavy chain
that meets the original interface of the CH3 domain of the other
heavy chain within the antibody, an amino acid residue is replaced
with an amino acid residue having a larger side chain volume,
thereby generating a protuberance within the interface of the CH3
domain of one heavy chain which is positionable in a cavity within
the interface of the CH3 domain of the other heavy chain and
[0508] b) the CH3 domain of the other heavy chain is altered, so
that within the original interface of the second CH3 domain that
meets the original interface of the first CH3 domain within the
antibody an amino acid residue is replaced with an amino acid
residue having a smaller side chain volume, thereby generating a
cavity within the interface of the second CH3 domain within which a
protuberance within the interface of the first CH3 domain is
positionable.
[0509] Said amino acid residue having a larger side chain volume
may optionally be selected from the group consisting of arginine
(R), phenylalanine (F), tyrosine (Y), tryptophan (W). Said amino
acid residue having a smaller side chain volume may optionally be
selected from the group consisting of alanine (A), serine (S),
threonine (T), valine (V).
[0510] Optionally, in some embodiments, both CH3 domains are
further altered by the introduction of cysteine (C) as amino acid
in the corresponding positions of each CH3 domain such that a
disulfide bridge between both CH3 domains can be formed.
[0511] The multispecific (e.g., biparatopic) antibodies of the
invention may comprise amino acid substitutions in Fab molecules
(including cross-Fab molecules) comprised therein which are
particularly efficient in reducing mispairing of light chains with
non-matching heavy chains (Bence-Jones-type side products), which
can occur in the production of Fab-based bi-/multispecific antigen
binding molecules with a VH/VL exchange in one (or more, in case of
molecules comprising more than two antigen-binding Fab molecules)
of their binding arms (see also PCT publication no. WO 2015/150447,
particularly the examples therein, incorporated herein by reference
in its entirety). The ratio of a desired multispecific antibodies
compared to undesired side products, in particular Bence Jones-type
side products occurring in one of their binding arms, can be
improved by the introduction of charged amino acids with opposite
charges at specific amino acid positions in the CH1 and CL domains
of a Fab molecule (sometimes referred to herein as "charge
modifications").
[0512] Therefore, in some embodiments, the antibodies of the
present invention comprising Fab molecules, comprises at least one
Fab with a heavy chain constant domain CH1 domain comprising charge
modifications as described herein, and a light chain constant CL
domain comprising charge modifications as described herein.
[0513] Charge modifications can be made either in the conventional
Fab molecule(s) comprised in the antibodies of the present
invention, or in the crossover Fab molecule(s) comprised in the
antibodies of the present invention (but not in both). In
particular embodiments, the charge modifications are made in the
conventional Fab molecule(s) comprised in the antibodies of the
present invention.
[0514] In some embodiments, in a Fab or cross-Fab comprising a
light chain constant domain CL comprising charge modifications and
a heavy chain constant domain CH1 comprising charge modifications,
charge modifications in the light chain constant domain CL are at
position 124 and optionally at position 123 (numbering according to
Kabat), and charge modifications in the heavy chain constant domain
CH1 are at position 147 and/or 213 (numbering according to Kabat EU
Index). In some embodiments, in the light chain constant domain CL
the amino acid at position 124 is substituted independently by
lysine (K), arginine (R) or histidine (H) (numbering according to
Kabat) (in one preferred embodiment independently by lysine (K)),
and in the heavy chain constant domain CH1 the amino acid at
position 147 and/or the amino acid at position 213 is substituted
independently by glutamic acid (E) or aspartic acid (D) (numbering
according to Kabat EU index.
H. Exemplary Antibodies
[0515] Aspects and embodiments concerning target binding (e.g.,
CEA-binding, FAP-binding or GPRC5D-binding) and aspects and
embodiments concerning DOTA binding can in some embodiments be
combined. That is, it may be preferred that the first and second
antibody each comprise a binding site for CEA, FAP or GPRC5D, e.g.,
comprising any of the sequences as described above, and that the
first and second antibodies associate to form a binding site for a
DOTA chelate having any of the sequences as described above. It is
also expressly contemplated that aspects and embodiments concerning
CEA binding, FAP or GPRC5D and/or DOTA binding can be combined with
preferred formats for the antibody as described above--i.e., in any
of the preferred formats, the part that binds the target antigen
may comprise CDRs or variable regions sequences as described above,
and/or the part that binds the radionuclide-labelled compound may
be a DOTA binder having CDRs and/or variable region sequences as
described above.
[0516] In one particular embodiment, the first antibody may
comprise:
[0517] a) a first full length antibody specifically binding to CEA
and consisting of two antibody heavy chains and two antibody light
chains; and
[0518] b) a polypeptide comprising or consisting of an antibody
heavy chain variable domain (VH) wherein the heavy chain variable
domain comprises heavy chain CDRs of SEQ ID NOs 35-37 (or wherein
CDR-H1 has the sequence GFSLTDYGVH (SEQ ID NO.: 148)), and/or
wherein the heavy chain variable domain has at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO 41;
[0519] wherein said polypeptide is fused with the N-terminus of the
VH domain, preferably via a peptide linker, to the C-terminus of
one of the two heavy chains of said first full-length antibody.
[0520] The first antibody does not comprise a light chain domain
which associates with the polypeptide of (b) to form a functional
binding domain for a radiolabelled compound.
[0521] It may be preferred that the polypeptide of (b) further
comprises one or more residues at the C-terminus of the VH domain,
e.g., 1-10 residues. Optionally, these may be one or more alanine
residues, optionally a single alanine residue. In another
embodiment, the additional residues may be an N-terminal portion of
the CH1 domain as described above, e.g., 1-10 residues from the
N-terminus of the CH1 domain, e.g., from the human IgG.sub.1 CH1
domain. For instance, the additional residues may be AST.
[0522] In some embodiments, the two antibody heavy chains in part
(a) have identical variable domains, optionally identical variable,
CH1 and/or CH2 domains. They may optionally differ only in their
CH3 domains, e.g., by the creation of knob into hole mutations and
other mutations intended to promote the correct association of
heterodimers.
[0523] The second antibody may comprise:
[0524] c) a second full length antibody specifically binding CEA
and consisting of two antibody heavy chains and two antibody light
chains; and
[0525] d) a polypeptide comprising or consisting of an antibody
light chain variable domain (VL) wherein the light chain variable
domain comprises CDRs of SEQ ID NO: 38-40 and/or wherein the light
chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100% identity to SEQ ID NO 42;
[0526] wherein said polypeptide is fused with the N-terminus of the
VL domain, preferably via a peptide linker, to the C-terminus of
one of the two heavy chains of said second full-length antibody and
wherein the second antibody does not comprise a heavy chain domain
which associates with the polypeptide of (d) to form a functional
binding domain for a radiolabelled compound.
[0527] In some embodiments, the two antibody heavy chains in part
(c) have identical variable domains to each other, optionally
identical variable, CH1 and/or CH2 domains. They may optionally
differ only in their CH3 domains, e.g., by the creation of knob
into hole mutations and other mutations intended to promote the
correct association of heterodimers.
[0528] The CEA-binding sites/sequences may be any of the
CEA-binding sites/sequences described above.
[0529] In one particular embodiment, the first antibody may have
CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody CH1A1A.
[0530] For example, the two light chains in (a) may comprise the
CDRs of SEQ ID Nos 22-24 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 26. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 103. In some embodiments, it may be preferred that the
two light chains in (a) are identical to each other.
[0531] The two antibody heavy chains in part (a) may comprise the
CDRs of SEQ ID NOs: 19-21 and/or the two antibody heavy chains in
part (a) comprise a variable domain having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO 25. In one
embodiment, one heavy chain in part (a) has the sequence of SEQ ID
NO: 100 and the other has the sequence of SEQ ID NO: 102.
[0532] In one specific embodiment, the first antibody may comprise
a first heavy chain of SEQ ID NO: 100, and second heavy chain of
SEQ ID NO: 101 (wherein the C-terminal AST is optional and may be
absent or substituted with anther C-terminal extension as described
herein) and a light chain of SEQ ID NO: 103.
[0533] The second antibody may also have CEA binding sequences
(i.e., CDRs or VH/VL domains) from the antibody CH1A1A.
[0534] For example, the two light chain in (c) may comprise the
CDRs of SEQ ID Nos 22-24 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 26. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 103. In some embodiments, it may be preferred that the
two light chains in (c) are identical to each other. In some
embodiments, it may be preferred that the two light chains in (c)
have the same sequence as the light chains in (a) of the first
antibody, e.g., that all said light chains in parts (a) and (c)
have the same sequence.
[0535] In some embodiments, the two antibody heavy chains in part
(c) comprise the CDRs of SEQ ID NOs: 19-21 and/or the two antibody
heavy chains in part (c) comprise a variable domain having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID
NO 25. In one embodiment, one heavy chain of part (c) has the
sequence of SEQ ID NO: 97 and the other has the sequence of SEQ ID
NO: 99.
[0536] In one specific embodiment, the second antibody may comprise
a first heavy chain of SEQ ID NO: 97, and second heavy chain of SEQ
ID NO: 98 and a light chain of SEQ ID NO: 103.
[0537] Similarly, aspects and embodiments concerning target binding
(e.g., CEA-binding, FAP-binding or GPRC5D-binding) and aspects and
embodiments concerning Pb-DOTAM binding can in some embodiments be
combined. That is, it may be preferred that the first and second
antibody each comprise a binding site for CEA, FAP or GPRC5D, e.g.,
comprising any of the sequences as described above, and that the
first and second antibodies associate to form a binding site for a
Pb-DOTAM chelate having any of the sequences as described above. It
is also expressly contemplated that aspects and embodiments
concerning CEA binding, FAP or GPRC5D and/or Pb-DOTAM binding can
be combined with preferred formats for the antibody as described
above--i.e., in any of the preferred formats, the part that binds
the target antigen may comprise CDRs or variable regions sequences
as described above, and/or the part that binds the
radionuclide-labelled compound may be a Pb-DOTAM binder having CDRs
and/or variable region sequences as described above.
[0538] In one particular embodiment, the first antibody may
comprise:
[0539] a) a first full length antibody specifically binding to CEA
and consisting of two antibody heavy chains and two antibody light
chains; and
[0540] b) a polypeptide comprising or consisting of an antibody
heavy chain variable domain (VH) wherein the heavy chain variable
domain comprises heavy chain CDRs of SEQ ID NOs 1-3, and/or wherein
the heavy chain variable domain has at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100% identity to SEQ ID NO 7;
[0541] wherein said polypeptide is fused with the N-terminus of the
VH domain, preferably via a peptide linker, to the C-terminus of
one of the two heavy chains of said first full-length antibody.
[0542] The first antibody does not comprise a light chain domain
which associates with the polypeptide of (b) to form a functional
binding domain for a radiolabelled compound.
[0543] It may be preferred that the polypeptide of (b) further
comprises one or more residues at the C-terminus of the VH domain,
optionally, one or more alanine residues, optionally a single
alanine residue. For example, the polypeptide of (b) may comprise
or consists of SEQ ID NO: 7 with a C-terminal alanine extension,
i.e., the sequence
TABLE-US-00008 (SEQ ID NO.: 150)
VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKA
LEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLTMTNMDP
VDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSSA.
[0544] In another embodiment, the additional residues may be an
N-terminal portion of the CH1 domain as described above, e.g., 1-10
residues from the N-terminus of the CH1 domain, e.g., from the
human IgG1 CH1 domain. For instance, the additional residues may be
AST.
[0545] In some embodiments, the two antibody heavy chains in part
(a) have identical variable domains, optionally identical variable,
CH1 and/or CH2 domains. They may optionally differ only in their
CH3 domains, e.g., by the creation of knob into hole mutations and
other mutations intended to promote the correct association of
heterodimers.
[0546] The second antibody may comprise:
[0547] c) a second full length antibody specifically binding CEA
and consisting of two antibody heavy chains and two antibody light
chains; and
[0548] d) a polypeptide comprising or consisting of an antibody
light chain variable domain (VL) wherein the light chain variable
domain comprises CDRs of SEQ ID NO: 4-6 and/or wherein the light
chain variable domain has at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100% identity to SEQ ID NO 8;
[0549] wherein said polypeptide is fused with the N-terminus of the
VL domain, preferably via a peptide linker, to the C-terminus of
one of the two heavy chains of said second full-length antibody and
wherein the second antibody does not comprise a heavy chain domain
which associates with the polypeptide of (d) to form a functional
binding domain for a radiolabelled compound.
[0550] In some embodiments, the two antibody heavy chains in part
(c) have identical variable domains to each other, optionally
identical variable, CH1 and/or CH2 domains. They may optionally
differ only in their CH3 domains, e.g., by the creation of knob
into hole mutations and other mutations intended to promote the
correct association of heterodimers.
[0551] In a particular embodiment, the first antibody may have CEA
binding sequences (i.e., CDRs or VH/VL domains) from the antibody
CH1A1A.
[0552] For example, the two light chains in (a) may comprise the
CDRs of SEQ ID Nos 22-24 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 26. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 34. In some embodiments, it may be preferred that the two
light chains in (a) are identical to each other.
[0553] The two antibody heavy chains in part (a) may comprise the
CDRs of SEQ ID NOs: 19-21 and/or the two antibody heavy chains in
part (a) comprise a variable domain having at least 90, 91, 92, 93,
94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO 25. In one
embodiment, one heavy chain in part (a) has the sequence of SEQ ID
NO: 27 and the other has the sequence of SEQ ID NO: 28.
[0554] In one specific embodiment, the first antibody may comprise
a first heavy chain of SEQ ID NO: 28, and second heavy chain of SEQ
ID NO: 32 (or a variant thereof comprising an additional C-terminal
alanine or other C-terminal extension as described herein, such as
an extension with AST) and a light chain of SEQ ID NO: 34. A
variant of SEQ ID NO: 32 with a C-terminal alanine extension is
shown below:
TABLE-US-00009 (SEQ ID NO.: 153)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGL
EWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDD
TAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVICFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCR
DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GGGGGSGGGGSGGGGSGGGGSVTLKESGPVLVKPTETLTLTCTVS
GFSLSTYSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTIS
KDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRG TLVTVSSA
[0555] In another particular embodiment, the first antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody A5B7 (including a humanized version thereof).
[0556] For example, the two light chains in (a) may comprise the
CDRs of SEQ ID Nos 46-48 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 50. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 54. In some embodiments, it may be preferred that the
two light chains in (a) are identical to each other.
[0557] In some embodiments, the two antibody heavy chains in part
(a) may comprise the CDRs of SEQ ID NOs: 43-45 and/or the two
antibody heavy chains in part (a) comprise a variable domain having
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 49. In one embodiment, one heavy chain in part (a) has
the sequence of SEQ ID NO: 51 and the other has the sequence of SEQ
ID NO: 53.
[0558] In one specific embodiment, the first antibody may comprise
a first heavy chain of SEQ ID NO: 51, and second heavy chain of SEQ
ID NO: 52 (or a variant thereof with a C-terminal alanine extension
or other C-terminal extension as described herein, such as an
extension with AST) and a light chain of SEQ ID NO: 54.
[0559] In another particular embodiment, the first antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody T84.66 (including a humanized version thereof).
[0560] For example, the two light chains in (a) may comprise the
CDRs of SEQ ID Nos 14-16 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 18. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 89. In some embodiments, it may be preferred that the
two light chains in (a) are identical to each other.
[0561] In some embodiments, the two antibody heavy chains in part
(a) may comprise the CDRs of SEQ ID NOs: 11-13 and/or the two
antibody heavy chains in part (a) comprise a variable domain having
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 17. In one embodiment, one heavy chain in part (a) has
the sequence of SEQ ID NO: 86 and the other has the sequence of SEQ
ID NO: 88.
[0562] In one specific embodiment, the first antibody may comprise
a first heavy chain of SEQ ID NO: 86, and second heavy chain of SEQ
ID NO: 87 (or a variant thereof in which the C-terminal "AST" is
absent or substituted by a different C-terminal extension as
disclosed herein) and a light chain of SEQ ID NO: 89.
[0563] In another particular embodiment, the first antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody 28A9 (including a humanized version thereof).
[0564] For example, the two light chains in (a) may comprise the
CDRs of SEQ ID Nos 62-64 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO: 66. In some embodiments they may have
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 96. In some embodiments, it may be preferred that the
two light chains in (a) are identical to each other.
[0565] In some embodiments, the two antibody heavy chains in part
(a) may comprise the CDRs of SEQ ID NOs: 59-61 and/or the two
antibody heavy chains in part (a) comprise a variable domain having
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 65. In one embodiment, one heavy chain in part (a) has
the sequence of SEQ ID NO: 93 and the other has the sequence of SEQ
ID NO: 95.
[0566] In one specific embodiment, the first antibody may comprise
a first heavy chain of SEQ ID NO: 93, and second heavy chain of SEQ
ID NO: 94 (or a variant thereof without the C-terminal "AST" or
with a different C-terminal extension as described herein) and a
light chain of SEQ ID NO: 96.
[0567] In some embodiments, the second antibody may have CEA
binding sequences (i.e., CDRs or VH/VL domains) from the antibody
CH1A1A.
[0568] For example, the two light chain in (c) may comprise the
CDRs of SEQ ID Nos 22-24 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 26. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 34. In some embodiments, it may be preferred that the two
light chains in (c) are identical to each other. In some
embodiments, it may be preferred that the two light chains in (c)
have the same sequence as the light chains in (a) of the first
antibody, e.g., that all said light chains in parts (a) and (c)
have the same sequence.
[0569] In some embodiments, the two antibody heavy chains in part
(c) comprise the CDRs of SEQ ID NOs: 19-21 and/or the two antibody
heavy chains in part (c) comprise a variable domain having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID
NO 25. In one embodiment, one heavy chain of part (c) has the
sequence of SEQ ID NO: 29 and the other has the sequence of SEQ ID
NO: 30.
[0570] In one specific embodiment, the second antibody may comprise
a first heavy chain of SEQ ID NO: 30, and second heavy chain of SEQ
ID NO: 33 and a light chain of SEQ ID NO: 34.
[0571] In another particular embodiment, the second antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from A5B7
(including a humanized version thereof).
[0572] For example, the two light chain in (c) may comprise the
CDRs of SEQ ID Nos 46-48 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 50. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 58. In some embodiments, it may be preferred that the two
light chains in (c) are identical to each other. In some
embodiments, it may be preferred that the two light chains in (c)
have the same sequence as the light chains in (a) of the first
antibody, e.g., that all said light chains in parts (a) and (c)
have the same sequence.
[0573] In some embodiments, the two antibody heavy chains in part
(c) comprise the CDRs of SEQ ID NOs: 43-45 and/or the two antibody
heavy chains in part (c) comprise a variable domain having at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to SEQ ID
NO 49. In one embodiment, one heavy chain of part (c) has the
sequence of SEQ ID NO: 55 and the other has the sequence of SEQ ID
NO: 57.
[0574] In one specific embodiment, the second antibody may comprise
a first heavy chain of SEQ ID NO: 55, and second heavy chain of SEQ
ID NO: 56 and a light chain of SEQ ID NO: 58.
[0575] In another particular embodiment, the second antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody T84.66 (including a humanized version thereof).
[0576] For example, the two light chains in (c) may comprise the
CDRs of SEQ ID Nos 14-16 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 18. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 89. In some embodiments, it may be preferred that the
two light chains in (c) are identical to each other.
[0577] In some embodiments, the two antibody heavy chains in part
(c) may comprise the CDRs of SEQ ID NOs: 11-13 and/or the two
antibody heavy chains in part (c) comprise a variable domain having
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 17. In one embodiment, one heavy chain in part (c) has
the sequence of SEQ ID NO: 83 and the other has the sequence of SEQ
ID NO: 85.
[0578] In one specific embodiment, the second antibody may comprise
a first heavy chain of SEQ ID NO: 83, and second heavy chain of SEQ
ID NO: 84 and a light chain of SEQ ID NO: 89.
[0579] In another particular embodiment, the second antibody may
have CEA binding sequences (i.e., CDRs or VH/VL domains) from the
antibody 28A9 (including a humanized version thereof).
[0580] For example, the two light chains in (c) may comprise the
CDRs of SEQ ID Nos 62-64 and/or may comprise light chains variable
domains having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to SEQ ID NO 66. In some embodiments they may have at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO: 96. In some embodiments, it may be preferred that the
two light chains in (c) are identical to each other.
[0581] In some embodiments, the two antibody heavy chains in part
(c) may comprise the CDRs of SEQ ID NOs: 59-61 and/or the two
antibody heavy chains in part (a) comprise a variable domain having
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to
SEQ ID NO 65. In one embodiment, one heavy chain in part (c) has
the sequence of SEQ ID NO: 90 and the other has the sequence of SEQ
ID NO: 92.
[0582] In one specific embodiment, the second antibody may comprise
a first heavy chain of SEQ ID NO: 90, and second heavy chain of SEQ
ID NO: 91 and a light chain of SEQ ID NO: 96.
[0583] In some embodiments, the first and the second antibody bind
the same epitope of CEA. Thus, for example, the first and the
second antibody may both have CEA binding sequences from the
antibody CH1A1A; or, the first and the second antibody may both
have CEA binding sequences from A5B7 (including a humanized version
thereof); or, the first and the second antibody may both have CEA
binding sequences from T84.66 (including a humanized version
thereof); or, the first and the second antibody may both have CEA
binding sequences from 28A9 (including a humanized version
thereof); or, the first and the second antibody may both have CEA
binding sequences from MFE23 (including a humanized version
thereof).
[0584] Thus, for example:
[0585] i) the first antibody may comprise a first heavy chain of
SEQ ID NO: 28, a second heavy chain of SEQ ID NO: 32 (optionally
with a C-terminal extension as described herein, e.g., AST) and a
light chain of SEQ ID NO: 34; and the second antibody may comprise
a first heavy chain of SEQ ID NO: 30, a second heavy chain of SEQ
ID NO: 33 and a light chain of SEQ ID NO: 34;
[0586] ii) the first antibody may comprise a first heavy chain of
SEQ ID NO: 51, a second heavy chain of SEQ ID NO: 52 (optionally
with a C-terminal extension as described herein, e.g., AST) and a
light chain of SEQ ID NO: 54; and the second antibody may comprise
a first heavy chain of SEQ ID NO: 55, a second heavy chain of SEQ
ID NO: 56 and a light chain of SEQ ID NO: 58;
[0587] iii) the first antibody may comprise a first heavy chain of
SEQ ID NO: 86, a second heavy chain of SEQ ID NO: 87 (wherein the
C-terminal AST residues are optional and may be absent or
substituted by an alternative C-terminal extension) and a light
chain of SEQ ID NO: 89; and the second antibody may comprise a
first heavy chain of SEQ ID NO: 83, a second heavy chain of SEQ ID
NO: 84 and a light chain of SEQ ID NO: 89; or
[0588] iv) the first antibody may comprise a first heavy chain of
SEQ ID NO: 93, a second heavy chain of SEQ ID NO: 94 (wherein the
C-terminal AST residues are optional and may be absent or
substituted by an alternative C-terminal extension) and a light
chain of SEQ ID NO: 96; and the second antibody may comprise a
first heavy chain of SEQ ID NO: 90, a second heavy chain of SEQ ID
NO: 91 and a light chain of SEQ ID NO: 96.
[0589] In other embodiments, the first and the second antibodies
bind to different epitopes of CEA, as discussed above. Thus, for
instance, the first antibody may have CEA binding sequences from
the antibody CH1A1A and the second antibody may have CEA binding
sequences from A5B7; or, the first antibody may have CEA binding
sequences from the antibody A5B7 and the second antibody may have
CEA binding sequences from CH1A1A. An example of the use of
bi-paratopic (CH1A1A and A5B7) pairs is described in Example
6c.
[0590] In still further specific embodiments, the target antigen
may be GPRC5D or FAP and the format may be as shown in FIG. 25B.
Optionally, the first and second antibody associate to form a
functional antigen binding site for a Pb-DOTAM chelate
(Pb-DOTAM).
[0591] Thus, in one embodiment (where the target antigen is
GPRC5D):
[0592] i) the first antibody comprises a first heavy chain of SEQ
ID NO: 104, a second heavy chain of SEQ ID NO:106 (wherein the
C-terminal alanine is optional and may be absent or replaced by an
alternative C-terminal extension as described herein) and a light
chain of SEQ ID NO: 107; and
[0593] ii) the second antibody comprises a first heavy chain of SEQ
ID NO: 104, a second heavy chain of SEQ ID NO: 105 and a light
chain of SEQ ID NO: 107.
[0594] In another embodiment (where the target antigen is FAP):
[0595] i) the first antibody comprises a first heavy chain of SEQ
ID NO: 108, a second heavy chain of SEQ ID NO:110 (wherein the
C-terminal alanine is optional and may be absent or replaced by an
alternative C-terminal extension as described herein) and a light
chain of SEQ ID NO: 111; and
[0596] ii) the second antibody comprises a first heavy chain of SEQ
ID NO: 108, a second heavy chain of SEQ ID NO:109 and a light chain
of SEQ ID NO: 111.
[0597] In still further specific embodiments, the target may be
CEA, e.g., having CEA binding sequences from the antibody CH1A1A,
and the format may be as shown in FIG. 25C. Optionally, the first
and second antibody associate to form a functional antigen binding
site for a Pb-DOTAM chelate (Pb-DOTAM). Thus, in one specific
embodiment:
[0598] i) the first antibody comprises a first heavy chain of SEQ
ID NO:112, a second heavy chain of SEQ ID NO: 114 and a light chain
of SEQ ID NO: 115; and
[0599] ii) the second antibody comprises a first heavy chain of SEQ
ID NO:112, a second heavy chain of SEQ ID NO: 113 and a light chain
of SEQ ID NO: 115.
I. Antibody Variants
[0600] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
Substitution, Insertion, and Deletion Variants
[0601] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs (CDRs) and FRs.
Conservative substitutions are shown in Table 1 under the heading
of "preferred substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or reduced or eliminated ADCC or CDC.
TABLE-US-00010 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile, Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0602] Amino acids may be grouped according to common side-chain
properties:
[0603] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0604] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0605] (3) acidic: Asp, Glu;
[0606] (4) basic: His, Lys, Arg;
[0607] (5) residues that influence chain orientation: Gly, Pro;
[0608] (6) aromatic: Trp, Tyr, Phe.
[0609] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0610] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more.
CDR residues are mutated and the variant antibodies displayed on
phage and screened for a particular biological activity (e.g.,
binding affinity).
[0611] Alterations (e.g., substitutions) may be made in CDRs, e.g.,
to improve antibody affinity. Such alterations may be made in CDR
"hotspots", i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mal. Biol. 207:179-196 (2008)), and/or residues
that contact antigen, with the resulting variant VH or VL being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
aspects of affinity maturation, diversity is introduced into the
variable genes chosen for maturation by any of a variety of methods
(e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves CDR-directed approaches, in which several CDR
residues (e.g., 4-6 residues at a time) are randomized. CDR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modelling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0612] In certain aspects, substitutions, insertions, or deletions
may occur within one or more CDRs so long as such alterations do
not substantially reduce the ability of the antibody to bind
antigen. For example, conservative alterations (e.g., conservative
substitutions as provided herein) that do not substantially reduce
binding affinity may be made in the CDRs. Such alterations may, for
example, be outside of antigen contacting residues in the CDRs. In
certain variant VH and VL sequences provided above, each CDR either
is unaltered, or contains no more than one, two or three amino acid
substitutions.
[0613] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex may be used to identify contact points
between the antibody and antigen. Such contact residues and
neighbouring residues may be targeted or eliminated as candidates
for substitution. Variants may be screened to determine whether
they contain the desired properties.
[0614] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme
prodrug therapy)) or a polypeptide which increases the serum
half-life of the antibody.
Glycosylation Variants
[0615] In certain aspects, an antibody provided herein is altered
to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0616] Where the antibody comprises an Fc region, the
oligosaccharide attached thereto may be altered. Native antibodies
produced by mammalian cells typically comprise a branched,
biantennary oligosaccharide that is generally attached by an
N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g.,
Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may
include various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some aspects, modifications of the oligosaccharide in
an antibody of the invention may be made in order to create
antibody variants with certain improved properties.
[0617] In one aspect, antibody variants are provided having a
non-fucosylated oligosaccharide, i.e. an oligosaccharide structure
that lacks fucose attached (directly or indirectly) to an Fc
region. Such non-fucosylated oligosaccharide (also referred to as
"afucosylated" oligosaccharide) particularly is an N-linked
oligosaccharide which lacks a fucose residue attached to the first
GlcNAc in the stem of the biantennary oligosaccharide structure. In
one aspect, antibody variants are provided having an increased
proportion of non-fucosylated oligosaccharides in the Fc region as
compared to a native or parent antibody. For example, the
proportion of non-fucosylated oligosaccharides may be at least
about 20%, at least about 40%, at least about 60%, at least about
80%, or even about 100% (i.e. no fucosylated oligosaccharides are
present). The percentage of non-fucosylated oligosaccharides is the
(average) amount of oligosaccharides lacking fucose residues,
relative to the sum of all oligosaccharides attached to Asn 297 (e.
g. complex, hybrid and high mannose structures) as measured by
MALDI-TOF mass spectrometry, as described in WO 2006/082515, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (EU numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
antibodies having an increased proportion of non-fucosylated
oligosaccharides in the Fc region may have improved Fc.gamma.RIIIa
receptor binding and/or improved effector function, in particular
improved ADCC function. See, e.g., US 2003/0157108; US
2004/0093621.
[0618] Examples of cell lines capable of producing antibodies with
reduced fucosylation include Lec13 CHO cells deficient in protein
fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545
(1986); US 2003/0157108; and WO 2004/056312, especially at Example
11), and knockout cell lines, such as alpha-1,6-fucosyltransferase
gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al.
Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol.
Bioeng., 94(4):680-688 (2006); and WO 2003/085107), or cells with
reduced or abolished activity of a GDP-fucose synthesis or
transporter protein (see, e.g., US2004259150, US2005031613,
US2004132140, US2004110282).
[0619] In a further aspect, antibody variants are provided with
bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide attached to the Fc region of the antibody is
bisected by GlcNAc. Such antibody variants may have reduced
fucosylation and/or improved ADCC function as described above.
Examples of such antibody variants are described, e.g., in Umana et
al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn
Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO
2003/011878.
[0620] Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087; WO 1998/58964; and
WO 1999/22764.
[0621] It may be preferred that the antibody is modified to reduce
the extent of glycosylation. In some embodiments the antibody may
be aglycosylated or deglycosylated. The antibody may include a
substitution at N297, e.g., N297D/A.
Fc Region Variants
[0622] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0623] In certain embodiments, the invention contemplates an
antibody variant with reduced effector function, e.g., reduced or
eliminated CDC, ADCC and/or Fc.gamma.R binding. In certain aspects,
the invention contemplates an antibody variant that possesses some
but not all effector functions, which make it a desirable candidate
for applications in which the half life of the antibody in vivo is
important yet certain effector functions (such as
complement-dependent cytotoxicity (CDC) and antibody-dependent
cell-mediated cytotoxicity (ADCC)) are unnecessary or
deleterious.
[0624] In vitro and/or in vivo cytotoxicity assays can be conducted
to confirm the reduction/depletion of CDC and/or ADCC activities.
For example, Fc receptor (FcR) binding assays can be conducted to
ensure that the antibody lacks Fc.gamma.R binding (hence likely
lacking ADCC activity), but retains FcRn binding ability. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.
9:457-492 (1991). Non-limiting examples of in vitro assays to
assess ADCC activity of a molecule of interest is described in U.S.
Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006); WO 2013/120929 A1).
[0625] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056), e.g., P329G. Such
Fc mutants include Fc mutants with substitutions at two or more of
amino acid positions 265, 269, 270, 297 and 327, including the
so-called "DANA" Fc mutant with substitution of residues 265 and
297 to alanine (U.S. Pat. No. 7,332,581).
[0626] In certain aspects, an antibody variant comprises an Fc
region with one or more amino acid substitutions which diminish
Fc.gamma.R binding, e.g., substitutions at positions 234 and 235 of
the Fc region (EU numbering of residues). In one aspect, the
substitutions are L234A and L235A (LALA). In certain aspects, the
antibody variant further comprises D265A and/or P329G in an Fc
region derived from a human IgG1 Fc region. In one aspect, the
substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region
derived from a human IgG1 Fc region. (See, e.g., WO 2012/130831).
In another aspect, the substitutions are L234A, L235A and D265A
(LALA-DA) in an Fc region derived from a human IgG1 Fc region.
[0627] In other embodiments, it may be possible to use a IgG
subtype with reduced effector function such as IgG4 or IgG2.
[0628] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0629] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished,
preferably diminished) C.sub.1q binding and/or Complement Dependent
Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551,
WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184
(2000).
[0630] In certain aspects, an antibody variant comprises an Fc
region with one or more amino acid substitutions, which reduce FcRn
binding, e.g., substitutions at positions 253, and/or 310, and/or
435 of the Fe-region (EU numbering of residues). In certain
aspects, the antibody variant comprises an Fc region with the amino
acid substitutions at positions 253, 310 and 435. In one aspect,
the substitutions are I253A, H310A and H435A in an Fe region
derived from a human IgG1 Fe-region. See, e.g., Grevys, A., et al.,
J. Immunol. 194 (2015) 5497-5508.
[0631] In certain aspects, an antibody variant comprises an Fc
region with one or more amino acid substitutions, which reduce FcRn
binding, e.g., substitutions at positions 310, and/or 433, and/or
436 of the Fc region (EU numbering of residues). In certain
aspects, the antibody variant comprises an Fc region with the amino
acid substitutions at positions 310, 433 and 436. In one aspect,
the substitutions are H310A, H433A and Y436A in an Fc region
derived from a human IgG1 Fc-region. (See, e.g., WO 2014/177460
A1). For instance, in some embodiments, normal FcRn binding may be
used.
[0632] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other
examples of Fc region variants.
[0633] The C-terminus of a heavy chain of the full-length antibody
as reported herein can be a complete C-terminus ending with the
amino acid residues PGK. The C-terminus of the heavy chain can be a
shortened C-terminus in which one or two of the C terminal amino
acid residues have been removed. The C-terminus of the heavy chain
may be a shortened C-terminus ending PG. In one aspect of all
aspects as reported herein, an antibody comprising a heavy chain
including a C-terminal CH3 domain, as specified herein, comprises a
C-terminal glycine residue (G446, EU index numbering of amino acid
positions). This is still explicitly encompassed with the term
"full length antibody" or "full length heavy chain" as used
herein.
Antibody Derivatives
[0634] In certain aspects, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
J. Recombinant Methods and Compositions
[0635] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, an isolated nucleic acid or a set of isolated nucleic
acids encoding a set of antibodies described herein is
provided.
[0636] For instance, a set of nucleic acids may comprise the
following nucleic acids encoding the first antibody: [0637] i) a
nucleic acid encoding a first heavy chain of the first antibody,
wherein said first heavy chain comprises a heavy chain of a full
length antibody specifically binding the target antigen, fused via
its C-terminus to a polypeptide comprising a VH domain of an
antigen binding site for the radiolabelled compound; [0638] ii) a
nucleic acid encoding a second heavy chain of the first antibody,
wherein said second heavy chain comprises a heavy chain of a full
length antibody specifically binding the target antigen and does
not comprise a VL domain of an antigen binding site for the
radiolabelled compound (optionally, the second heavy chain consists
of the heavy chain of a full length antibody specifically binding
the target antigen); [0639] iii) a nucleic acid encoding the light
chain of the first antibody. A set of nucleic acids according to
the invention may additionally or alternatively comprise the
following nucleic acids encoding the second antibody: [0640] iv) a
nucleic acid encoding a first heavy chain of the second antibody,
wherein said first heavy chain comprises a heavy chain of a full
length antibody specifically binding the target antigen, fused via
its C-terminus to a polypeptide comprising a VL domain of an
antigen binding site for the radiolabelled compound; [0641] v) a
nucleic acid encoding a second heavy chain of the second antibody,
wherein said second heavy chain comprises a heavy chain of a full
length antibody specifically binding the target antigen and does
not comprise a VH domain of an antigen binding site for the
radiolabelled compound (optionally, the second heavy chain consists
of the heavy chain of a full length antibody specifically binding
the target antigen); [0642] vi) a nucleic acid encoding the light
chain of the second antibody.
[0643] In some embodiments, certain of these nucleic acids may be
the same as each other. For instance, the nucleic acid in (iii) may
the same as in (vi), such that the overall set comprises only 5
distinct nucleic acid sequences.
[0644] The nucleic acids can be comprised in one or more nucleic
acid molecules or expression vectors.
[0645] Thus, in a further embodiment, one or more vectors (e.g.,
expression vectors) comprising such nucleic acid(s) are provided.
In one embodiment, each respective heavy and light chain is
expressed from an individual plasmid.
[0646] In a further embodiment, a host cell or a set of host cells
comprising such nucleic acid(s) or vector(s) is provided. In one
embodiment, a first host cell is provided expressing the first
antibody, and a second host cell is provided expressing the second
antibody.
[0647] In one such embodiment, a first host cell comprises (e.g.,
has been transformed with): (1) a vector comprising nucleic acids
(i)-(iii) above, or (2) a first vector comprising nucleic acid (i),
a second vector comprising nucleic acid (ii) and a third vector
comprising nucleic acid (iii); or (3) two vectors which
collectively comprise nucleic acids (i)-(iii) above. A second host
cell comprises (e.g., has been transformed with): (1) a vector
comprising nucleic acids (iv)-(vi) above, or (2) a first vector
comprising nucleic acid (iv), a second vector comprising nucleic
acid (v) and a third vector comprising nucleic acid (vi); or (3)
two vectors which collective comprise nucleic acids (iv)-(vi)
above.
[0648] In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an antibody
according to the invention is provided, wherein the method
comprises culturing a host cell comprising a nucleic acid encoding
the antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0649] For recombinant production of an antibody, nucleic acid
encoding an antibody, e.g., as described above, is isolated and
inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0650] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, K. A., In: Methods in
Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press,
Totowa, N.J. (2003), pp. 245-254, describing expression of antibody
fragments in E. coli.) After expression, the antibody may be
isolated from the bacterial cell paste in a soluble fraction and
can be further purified.
[0651] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized", resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22
(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006)
210-215.
[0652] Suitable host cells for the expression of (glycosylated)
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0653] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0654] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293T cells as described, e.g., in Graham, F. L.
et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells
(BHK); mouse sertoli cells (TM4 cells as described, e.g., in
Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney
cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo
rat liver cells (BRL 3A); human lung cells (W138); human liver
cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as
described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci.
383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl.
Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as
Y0, NS0 and Sp2/0. For a review of certain mammalian host cell
lines suitable for antibody production, see, e.g., Yazaki, P. and
Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster
Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
K. Assays
[0655] Antibodies provided herein may be identified, screened for,
or characterized for their physical/chemical properties and/or
biological activities by various assays known in the art.
[0656] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc.
Antibody Affinity
[0657] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) for the target antigen of .ltoreq.1
.mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g., 10.sup.-8 M or less,
e.g., from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M), or as otherwise stated herein.
[0658] In certain embodiments, an antigen binding site for the
radiolabelled compound has a dissociation constant (Kd) for the
radiolabelled compound of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g., 10-8 M or less, e.g., from 10-8 M to 10-13
M, e.g., from 10-9 M to 10-13 M). In some embodiments, the Kd is 1
nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM
or less, 20 pM or less, 10 pM or less, 5 pM or less or 1 pM or
less, or as otherwise stated herein. For instance, the functional
binding site may bind the radiolabelled compound/metal chelate with
a Kd of about 1 pM-1 nM, e.g., about 1-10 pM, 1-100 pM, 5-50 pM,
100-500 pM or 500 pM-1 nM.
[0659] In one embodiment, Kd is measured by a radiolabelled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labelled antigen in the presence of a titration series
of unlabelled antigen, then capturing bound antigen with an
anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.
Biol. 293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20) in PBS. When the plates have dried, 150
.mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is added,
and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0660] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CM5 chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/m11 (.about.0.2 .mu.M) before injection at a flow rate
of 5 .mu.l/minute to achieve approximately 10 response units (RU)
of coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.offl.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10.sup.6
M.sup.-1 s.sup.-1 by the surface plasmon resonance assay above,
then the on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0661] In another embodiment, Kd is measured using a SET (solution
equilibration titration) assay. According to this assay, test
antibodies are typically applied in a constant concentration and
mixed with serial dilutions of the test antigen. After incubation
to establish an equilibrium, the portion of free antibodies is
captured on an antigen coated surface and detected with
labelled/tagged anti-species antibody, generally using
electochemiluminescence (e.g., as described in Haenel et al
Analytical Biochemistry 339 (2005) 182-184).
[0662] For example, in one embodiment 384-well streptavidin plates
(Nunc, Microcoat #11974998001) are incubated overnight at 4.degree.
C. with 25 .mu.l/well of an antigen-Biotin-Isomer Mix in PBS-buffer
at a concentration of 20 ng/ml. For equilibration of antibody
samples with free antigen: 0.01 nM-1 nM of antibody is titrated
with the relevant antigen in 1:3, 1:2 or 1:1.7 dilution steps
starting at a concentration of 2500 nM, 500 nM or 100 nM of
antigen. The samples are incubated at 4.degree. C. overnight in
sealed REMP Storage polypropylene microplates (Brooks). After
overnight incubation, streptavidin plates are washed 3.times. with
90 .mu.l PBST per well. 15 .mu.l of each sample from the
equilibration plate is transferred to the assay plate and incubated
for 15 min at RT, followed by 3.times.90 .mu.l washing steps with
PBST buffer. Detection is carried out by adding 25 .mu.l of a goat
anti-human IgG antibody-POD conjugate (Jackson, 109-036-088, 1:4000
in OSEP), followed by 6.times.90 .mu.l washing steps with PBST
buffer. 25 .mu.l of TMB substrate (Roche Diagnostics GmbH, Cat.
No.: 11835033001) are added to each well. Measurement takes place
at 370/492 nm on a Safire2 reader (Tecan).
[0663] In another embodiment, Kd is measured using a KinExA
(kinetic exclusion) assay. According to this assay, the antigen is
typically titrated into a constant concentration of antibody
binding sites, the samples are allowed to equilibrate, and then
drawn quickly through a flow cell where free antibody binding sites
are captured on antigen-coated beads, while the antigen-saturated
antibody complex is washed away. The bead-captured antibody is then
detected with a labelled anti-species antibody, e.g., fluorescently
labelled (Bee et al PloS One, 2012; 7 (4): e36261). For example, in
one embodiment, KinExA experiments are performed at room
temperature (RT) using PBS pH 7.4 as running buffer. Samples are
prepared in running buffer supplemented with 1 mg/ml BSA ("sample
buffer"). A flow rate of 0.25 ml/min is used. A constant amount of
antibody with 5 pM binding site concentration is titrated with
antigen by twofold serial dilution starting at 100 pM
(concentration range 0.049 pM-100 pM). One sample of antibody
without antigen serves as 100% signal (i.e. without inhibition).
Antigen-antibody complexes are incubated at RT for at least 24 h to
allow equilibrium to be reached. Equilibrated mixtures are then
drawn through a column of antigen-coupled beads in the KinExA
system at a volume of 5 ml permitting unbound antibody to be
captured by the beads without perturbing the equilibrium state of
the solution. Captured antibody is detected using 250 ng/ml Dylight
650.RTM.-conjugated anti-human Fc-fragment specific secondary
antibody in sample buffer. Each sample is measured in duplicates
for all equilibrium experiments. The KD is obtained from non-linear
regression analysis of the data using a one-site homogeneous
binding model contained within the KinExA software (Version 4.0.11)
using the "standard analysis" method.
L. Therapeutic Methods and Compositions
[0664] The set of antibodies as described herein may be used in
therapeutic methods. In one aspect a set of antibodies as described
herein is provided for use as a medicament. In certain aspects, a
set of antibodies for use in a method of treatment is provided.
[0665] As discussed above, in some aspects, sets of antibodies
according to the present invention are suitable for any treatment
in which it is desired to deliver a radionuclide to a target cell
in a subject. For example, there is provided a set of antibodies as
described herein for use in a method of pre-targeted
radioimmunotherapy, e.g., for cancer treatment.
[0666] In certain aspects, the invention provides the set of
antibodies for use in a method of pre-targeted radioimmunotherapy
in an individual comprising administering to the individual an
effective amount of the set of antibodies. An "individual"
according to any of the above aspects is preferably a human.
[0667] As noted above, the treatment may be of any condition that
is treatable by cytotoxic activity targeted to diseased cells of
the patient. The treatment is preferably of a tumour or cancer.
However, the applicability of the invention is not limited to
tumours and cancers. For example, the treatment may also be of
viral infection, or infection by another pathogenic organism, e.g.,
a prokaryote. Optionally, targeting may also be to T-cells for
treatment of T-cell driven autoimmune disease or T-cell blood
cancers. Thus, conditions to be treated may include viral
infections such as HIV, rabies, EBV and Kaposi's sarcoma-associated
herpesvirus, and autoimmune diseases such as multiple sclerosis and
graft-versus-host disease drugs.
[0668] The term "cancer" as used herein include both solid and
hematologic cancers, such as lymphomas, lymphocytic leukemias, lung
cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell
lung cancer, bone cancer, pancreatic cancer including pancreatic
ductal adenocarcinoma (PDAC), skin cancer, cancer of the head or
neck, cutaneous or intraocular melanoma, uterine cancer, ovarian
cancer, cancer of the anal region, stomach cancer, gastric cancer,
colorectal cancer, which may be colon cancer and/or rectal cancer,
breast cancer, uterine cancer, carcinoma of the fallopian tubes,
carcinoma of the endometrium, carcinoma of the cervix, carcinoma of
the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of
the esophagus, cancer of the small intestine, cancer of the
endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft
tissue, cancer of the urethra, cancer of the penis, prostate
cancer, cancer of the bladder, cancer of the kidney or ureter,
renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,
hepatocellular cancer, biliary cancer, neoplasms of the central
nervous system (CNS), spinal axis tumours, brain stem glioma,
glioblastoma multiforme, astrocytomas, schwanomas, ependymomas,
medulloblastomas, meningiomas, squamous cell carcinomas, pituitary
adenoma and Ewings sarcoma, including refractory versions of any of
the above cancers, checkpoint-inhibitor experienced versions of any
of the above cancers, or a combination of one or more of the above
cancers.
[0669] A method of targeting a radioisotope to a cell, tissue or
organ for therapy may comprise: [0670] i) administering to the
subject a first and a second antibody as described herein
(simultaneously or sequentially, in either order), wherein the
antibodies bind to the target antigen and localise to the surface
of a cell expressing the target antigen; and wherein association of
the first and second antibody forms a functional binding site for
the radiolabelled compound; [0671] and [0672] ii) subsequently
administering a radiolabelled compound, wherein the radiolabelled
compound binds to functional binding site for the radiolabelled
compound.
[0673] The radiolabelled compound is labelled with a radioisotope
which is cytotoxic to cells. Suitable radioisotopes include alpha
and beta emitters as discussed above.
[0674] In methods of pre-targeted radioimmunotherapy which make use
of a bispecific antibody (i.e., not a "split" antibody according to
the present invention) it is common practice to administer a
clearing agent or a blocking agent, between administration of the
antibody and administration of the radiolabelled compound. Clearing
agents bind to the antibodies and enhance their rate of clearance
from the body. They include anti-idiotype antibodies. Blocking
agents are typically agents which bind to the antigen binding site
for the radiolabelled compound, but which are not themselves
radiolabelled. For example, where the radiolabelled compound
comprises a chelator loaded with a radioisotope of a certain
chemical element (e.g., a metal), the blocking agent may comprise
the same chelator loaded with a non-radioactive isotope of the same
element (e.g., metal), or may comprise a non-loaded chelator or a
chelator loaded with a different non-radioactive moiety (e.g., a
non-radioactive isotope of a different element), provided that it
can still be bound by the antigen-binding site. It some cases, the
blocking agent may additionally comprise a moiety which increases
the size and/or hydrodynamic radius of the molecule. These hinder
the ability of the molecule to access the tumour, without
interfering with the ability of the molecule to bind to the
antibody in the circulation. Exemplary moieties include hydrophilic
polymers. The moiety may be a polymer or co-polymer e.g., of
dextran, dextrin, PEG, polysialic acids (PSAs), hyaluronic acid,
hydroxyethyl-starch (HES) or poly(2-ethyl 2-oxazoline) (PEOZ). In
other embodiments the moiety may be a non-structured peptide or
protein such as XTEN polypeptides (unstructured hydrophilic protein
polymers), homo-amino acid polymer (HAP), proline-alanine-serine
polymer (PAS), elastin-like peptide (ELP), or gelatin-like protein
(GLK). Further exemplary moieties include proteins such as albumin
e.g., bovine serum albumin, or IgG. Suitable molecular weights for
the moieties/polymers may be in the range e.g., of at least 50 kDa,
for example between 50 kDa to 2000 kDa. For example, the molecular
weight may be 200-800 kDa, optionally greater than 300, 350, 400 or
450 kDa, and optionally less than 700, 650, 600 or 550 kDa,
optionally about 500 kDa.
[0675] According to certain aspects of the present invention, there
is no step of administering a clearing agent or a blocking agent to
the subject. In certain aspects, there is no step of administering
any agent which binds to the first or the second antibody, between
the administration of the antibodies and the administration of the
radiolabelled compound. In certain aspects, there is no step of
administering any agent between the administration of the
antibodies and the radiolabelled compound, except optionally a
compound selected from a chemotherapeutic agent, immunotherapeutic
and a radiosensitizer. In some embodiments, no agent is
administered between the administration of the antibodies and the
administration of the radiolabelled compound. In some embodiments
there may be no injection or infusion of any other agent to the
subject, between the administration of the antibody and the
administration of the radiolabelled compound.
[0676] In some embodiments, the method may be a two-step method of
pre-targeted radioimmunotherapy consisting or consisting
essentially of the steps of i) administering the set of antibodies
(wherein the first and second antibody may be administered
simultaneously or sequentially in either order) and ii)
subsequently administering the radiolabelled compound. The
treatment may involve multiple cycles of such therapy, i.e.,
multiple cycles of these two steps. An exemplary treatment cycle
duration is 28 days, in which the set of antibodies is administered
on day 1 of the cycle, and the radiolabelled compound is optionally
administered on day 1, 2, 3, 4, 5, 6, 7, or 8 of the cycle, e.g.,
on day 7. The number of therapeutic cycles may vary. In one
embodiment, there may be 4, 5, or 6 treatment cycles.
[0677] The present inventors have surprisingly determined that
using antibodies according to the invention, it is possible to
obtain therapeutically effective uptake of the radiolabelled
compound into the tumour, while avoiding excessive accumulation of
radioactivity in normal tissues. Indeed, in the examples the level
of accumulation of radioactivity in non-target tissues was found to
be lower than in a three-step PRIT method, using bispecific
antibodies and a clearing step, while also making use of a simpler
procedure.
[0678] In some embodiments, the radiolabelled compound may be
administered to the subject once the first and second antibody have
been given a suitable period of time to localise to the target
cells. For instance, in some embodiments, the radiolabelled
compound may be administered to the subject immediately after the
first and second antibodies or at least 4 hours, 8 hours, 1 day, or
2 days, after the first and second antibodies. Optionally, it may
be administered no more than 3 days, 5 days, or 7 days after the
first and second antibodies. In one particular embodiment, the
radiolabelled compound may be administered to the subject 2 to 7
days after the first and second antibodies.
[0679] In some embodiments, the antibodies described herein may be
administered as part of a combination therapy. For example, they
may be administered in combination with one or more
chemotherapeutic agents: the chemotherapeutic agent and the
antibody may be administered simultaneously or sequentially, in
either order. Additionally or alternatively, they may be
administered in combination with one or more immunotherapeutic: the
immunotherapeutic agent and the antibody may be administered
simultaneously or sequentially, in either order.
[0680] In some embodiments, the antibodies described herein may
additionally or alternatively be administered in combination with
radiosensitizers. The radiosensitizer and the antibody may be
administered simultaneously or sequentially, in either order.
[0681] Antibodies of the invention (and any additional therapeutic
agent, e.g., the radiolabelled compound) can be administered by any
suitable means, including parenteral, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. Dosing can be by any suitable route, e.g., by
injections, such as intravenous or subcutaneous injections.
[0682] In some embodiments, one or more dosimetry cycles may be
used prior to one or more treatment cycles as described above. A
dosimetry cycle may comprise the steps of i) administering the set
of antibodies (wherein the first and second antibody may be
administered simultaneously or sequentially in either order) and
ii) subsequently administering a compound suitable for imaging
radiolabelled with a gamma-emitter (wherein said radiolabelled
compound binds to functional binding site for the radiolabelled
compound). The compound may be the same as the compound used in the
subsequent treatment cycles, except that it is labelled with a
gamma emitter rather than an alpha or beta emitter. For example, in
one embodiment, the radiolabelled compound used in the dosimetry
cycle may be .sup.203Pb-DOTAM and the radiolabelled compound used
in the treatment cycle may be .sup.212Pb-DOTAM. The patient may be
subject to imaging to determine the uptake of the compound into the
tumour and/or to estimate the absorbed dose of the compound. This
information may be used to estimate the expected radiation exposure
in subsequent treatment steps and to adjust the dose of the
radiolabelled compound used in the treatment steps to a safe
level.
M. Pharmaceutical Formulations
[0683] The first and second antibody described herein may be
formulated in a single pharmaceutical composition or in separate
pharmaceutical compositions. Thus, in a further aspect, the present
invention provides a pharmaceutical composition comprising the
first and second antibodies of the invention, or a first
pharmaceutical formulation comprising the first antibody of the
invention and a second pharmaceutical composition comprising the
second antibody of the invention, e.g., for use in any of the
therapeutic or diagnostic methods described herein. In one
embodiment, the pharmaceutical composition further comprises a
pharmaceutically acceptable carrier. In another embodiment, the
pharmaceutical composition further comprises at least one
additional therapeutic agent, e.g., as described below.
[0684] Pharmaceutical formulations of antibodies as described
herein may be prepared by mixing such antibody having the desired
degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions.
[0685] Pharmaceutically acceptable carriers are generally nontoxic
to recipients at the dosages and concentrations employed, and
include, but are not limited to: buffers such as histidine,
phosphate, citrate, acetate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride; benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Halozyme, Inc.).
Certain exemplary sHASEGPs and methods of use, including rHuPH20,
are described in US Patent Publication Nos. 2005/0260186 and
2006/0104968. In one aspect, a sHASEGP is combined with one or more
additional glycosaminoglycanases such as chondroitinases.
[0686] Exemplary lyophilized antibody compositions are described in
U.S. Pat. No. 6,267,958. Aqueous antibody compositions include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter compositions including a histidine-acetate buffer.
[0687] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide chemotherapeutic agents, immunotherapeutic agents
and/or radiosensitizers as discussed above. Such active ingredients
are suitably present in combination in amounts that are effective
for the purpose intended.
[0688] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0689] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0690] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
N. Methods and Compositions for Diagnosis and Detection
[0691] The set of antibodies as described herein may also be used
in methods of diagnosis or imaging, preferably methods of or
comprising pre-targeted radioimmunoimaging. Accordingly, the
present invention provides methods of diagnosis and imaging. It
further provides use of the set of antibodies in a method of
imaging as described herein, and a set of antibodies as described
herein (i.e., the first and second antibody as described herein)
for use in a method of diagnosis carried out on a subject, e.g., on
the human or animal body.
[0692] The imaging methods are suitable for imaging the presence
and/or distribution of the target antigen in the body. For example,
the method may be a method of imaging cells expressing an antigen
associated with a disease, such as any of the disease conditions
discussed above. Optionally the method is for imaging tumours or
cancer. The method may be for the purpose of diagnosing a subject
suspected of having a proliferative disorder such as cancer, or an
infectious disease.
[0693] In some embodiments it may be preferred that the subject is
a human.
[0694] A method of targeting a radioisotope to a tissue or organ
for imaging or diagnosis may comprise: [0695] i) administering to
the subject a first and a second antibody as described herein
(simultaneously or sequentially, in either order), wherein the
antibodies bind to a target antigen and localise to the surface of
a cell expressing the target antigen, wherein association of the
first and second antibody forms a functional binding site for the
radiolabelled compound; [0696] and [0697] ii) subsequently
administering a radiolabelled compound, wherein the radiolabelled
compound binds to the functional binding site for the radiolabelled
compound.
[0698] Optionally, the method may further comprise: [0699] iii)
imaging the tissue or organ where the radiolabelled compound has
localised, or is expected to be localised.
[0700] Optionally, the method may further comprise one or more
steps of forming a diagnosis, delivering a diagnosis to the
subject, and/or determining and/or administering a suitable
treatment on the basis of the diagnosis.
[0701] In another embodiment, a method of the invention may
comprise imaging a tissue or organ of a subject, wherein the
subject has been previously administered with: [0702] i) a first
and a second antibody as described herein (simultaneously or
sequentially, in either order), wherein the antibodies bind to a
target antigen and localise to the surface of a cell expressing the
target antigen, and wherein association of the first and second
antibody forms a functional binding site for the radiolabelled
compound; and [0703] ii) a radiolabelled compound, wherein the
radiolabelled compound binds to the antigen binding site for said
radiolabelled compound formed by association of the first and
second antibody.
[0704] In imaging and/or diagnostic methods as described herein,
the radiolabelled compound is labelled with a radioisotope which is
suitable for imaging. Suitable radioisotopes include gamma emitters
as discussed above.
[0705] In conventional methods of pre-targeted radioimaging, it is
common practice to administer a clearing or blocking agent between
the administration of the antibody and the administration of the
radiolabelled compound, e.g., a clearing or blocking agent as
described above.
[0706] In certain embodiments of the present invention, there is no
step of administering a clearing agent or a blocking agent. In
certain aspects, there is no step of administering any agent which
binds to the first or the second antibody, between the
administration of the antibodies and the administration of the
radiolabelled compound. In certain aspects, there is no step of
administering any agent between the administration of the
antibodies and the radiolabelled compound, except optionally a
compound selected from a chemotherapeutic agent, immunotherapeutic
agent and a radiosensitizer. In some embodiments, no agent is
administered between the administration of the antibodies and the
administration of the radiolabelled compound. In some embodiments
there may be no injection or infusion of any other agent to the
subject, between the administration of the antibody and the
administration of the radiolabelled compound.
[0707] In some embodiments, the radiolabelled compound may be
administered to the subject once the first and second antibody have
been given a suitable period of time to localise to the target
cells. For instance, in some embodiments, the radiolabelled
compound may be administered to the subject immediately after the
first and second antibodies or at least 4 hours, 8 hours, 1 day, or
2 days after the first and second antibodies. Optionally, it may be
administered no more than 3 days, 5 days, or 7 days after the first
and second antibodies. In one particular embodiment, the
radiolabelled compound may be administered to the subject 2 to 7
days after the first and second antibodies.
[0708] In some embodiments, the imaging method may be a method of
pre-targeted radioimaging consisting or consisting essentially of
the steps of i) administering the set of antibodies (wherein the
first and second antibody may be administered simultaneously or
sequentially in either order) ii) subsequently administering the
radiolabelled compound and iii) imaging the tissue or organ of
interest. A diagnostic method may consist or consist essentially of
said steps followed by steps of forming a diagnosis, which may then
be delivered to the patient and may be used as the basis for
selected and/or administering a treatment regimen.
[0709] The target antigen may be any target antigen as discussed
herein. In some embodiments, the target antigen may be a
tumour-specific antigen as discussed above, and the imaging may be
a method of imaging a tumour or tumours. The individual may be
known to or suspected of having a tumour.
[0710] For example, the method may be a method of imaging tumours
in an individual having or suspected of having lung cancer, non
small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer,
bone cancer, pancreatic cancer including PDAC, skin cancer, cancer
of the head or neck, cutaneous or intraocular melanoma, uterine
cancer, ovarian cancer, colorectal cancer which may be rectal
cancer and/or colon cancer, cancer of the anal region, stomach
cancer, gastric cancer, breast cancer, uterine cancer, carcinoma of
the fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine system, cancer of the thyroid gland, cancer
of the parathyroid gland, cancer of the adrenal gland, sarcoma of
soft tissue, cancer of the urethra, cancer of the penis, prostate
cancer, cancer of the bladder, cancer of the kidney or ureter,
renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,
hepatocellular cancer, biliary cancer, neoplasms of the central
nervous system (CNS), spinal axis tumours, brain stem glioma,
glioblastoma multiforme, astrocytomas, schwanomas, ependymomas,
medulloblastomas, meningiomas, squamous cell carcinomas, pituitary
adenoma and Ewings sarcoma, including refractory versions of any of
the above cancers, or check-point inhibitor experienced versions of
any of these cancers, or a combination of one or more of the above
cancers.
III. Sequences
TABLE-US-00011 [0711] SEQ ID NO Description Sequence 1 heavy chain
gfslstysms CDR1, <Pb- Dotam> 2 heavy chain figsrgdtyyaswakg
CDR2 <Pb- Dotam> 3 heavy chain erdpygggaypphl CDR3 <Pb-
Dotam> 4 light chain qsshsvysdndla CDR1, <Pb-Dotam> 5
light chain qasklas CDR2 <Pb-Dotam> 6 light chain
lggyddesdtyg CDR3 <Pb-Dotam> 7 heavy chain vtlkesgpvl
vkptetltlt variable domain ctvsgfslst ysmswirqpp gkalewlgfi
gsrgdtyyas wakgrltisk dtsksqwlt <Pb-Dotam> mtnmdpvdta
tyycarerdp PRIT-0213 ygggaypphl wgrgtlvtvs s 8 light chain
iqmtqspssl sasvgdrvti variable domain tcqsshsvys dndlawyqqk
<Pb-Dotam> pgkapklliy qasklasgvp PRIT-0213 srfsgsgsgt
dftltisslq pedfatyycl ggyddesdty gfgggtkvei k 9 heavy chain
vqlqqwgagl Ikpsetlslt variable domain cavygfslst ysmswirqpp
<Pb-Dotam> gkglewigfi gsrgdtyyas PRIT-0214 wakgrvtisr
dtsknqvslk lssvtaadta vyycarerdp ygggaypphl wgrgtlvtvs s 10 light
chain iqmtqspssl sasvgdrvti variable domain tcqsshsvys dndlawyqqk
<Pb-Dotam> pgkapklliy qasklasgvp PRIT-0214 srfsgsgsgt
dftltisslq pedfatyycl ggyddesdty gfgggtkvei k 11 heavy chain
GFNIKDTYMH CDR1 <CEA> T84.66 12 heavy chain RIDPANGNSKYVPKFQG
CDR2 <CEA> T84.66 13 heavy chain FGYYVSDYAMAY CDR3
<CEA> T84.66 14 light chain CDR1 RAGESVDIFGVGFLH <CEA>
T84.66 15 light chain CDR2 RASNRAT <CEA> T84.66 16 light
chain CDR3 QQTNEDPYT <CEA> T84.66 17 heavy chain
qvqlvqsgaevkkpgssvkvsc variable domain kasgfnikdtymhwvrqapgqg
<CEA> T84.66 lewmgridpangnskyvpkfqg rvtitadtststaymelsslrs
edtavyycapfgyyvsdyamay wgqgtlvtvss 18 light chain
eivltqspatlslspgeratls variable domain cragesvdifgvgflhwyqqkp
<CEA> T84.66 gqaprlliyrasnratgiparf sgsgsgtdftltisslcpcdfa
vyycqqtncdpytfgqgtklei k 19 heavy chain GYTFTEFGMN CDR1 <CEA>
CH1A1A 20 heavy chain WINTKTGEATYVEEFKG CDR2 <CEA> CH1A1A 21
heavy chain WDFAYYVEAMDY CDR3 <CEA> CH1A1A 22 light chain
KASAAVGTYVA CDR1 <CEA> CH1A1A 23 light chain SASYRKR CDR2
<CEA> CH1A1A 24 light chain HQYYTYPLFT CDR3 <CEA>
CH1A1A 25 heavy chain QVQLVQSGAEVKKPGASVKVSCKAS variable
GYTFTEFGMNWVRQAPGQGLEWMGW domain INTKTGEATYVEEFKGRVTFTTDTS
<CEA> TSTAYMELRSLRSDDTAVYYCARWD CH1A1A FAYYVEAMDYWGQGTTVTVSS
26 light chain DIQMTQSPSSLSASVGDRVTITCKA variable
SAAVGTYVAWYQQKPGKAPKLLIYS domain ASYRKRGVPSRFSGSGSGTDFTLTI
<CEA> SSLQPEDFATYYCHQYYTYPLFTFG CH1A1A QGTKLEIK 27 Heavy
chain QVQUVQSGAEVKKPGASVKVSCKAS <CEA> of
GYTFTEFGMNWVRQAPGQGLEWMGW P1AD8749 RNTKTGEATYVEEFKGRVTFTTDTS
without TSTAYMELRSLRSDDTAVYYCARWD linker and
FAYYVEAMDYWGQGTTVTVSSASTK <DOTAM- GPSVFPLAPSSKSTSGGTAALGCLV
VH>.fwdarw. Same KDYFPEPVTVSWNSGALTSGVHTFP Plasmid as
AVLQSSGLYSLSSVVTVPSSSLGTQ SeqID32, TYICNVNHKPSNTKVDKKVEPKSCD
lacking KTHTCPPCPAPEAAGGPSVFLFPPK linker and
PKDTLMISRTPEVTCVVVDVSHEDP <DOTAM> EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 28 P1AD8749
QVQLVQSGAEVKKPGASVKVSCKAS Heavy GYTFTEFGMNWVRQAPGQGLEWMGW chain
hole INTKTGEATYVEEFKGRVTFTTDTS <CEA>
TSTAYMELRSLRSDDTAVYYCARWD CH1A1A FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDSFFLVSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG 29 Heavy chain QVQLVQSGAEVKKPGASVKVSCKAS
<CEA> of GYTFTEFGMNWVRQAPGQGLEWMGW P1AD8592
INTKTGEATYVEEFKGRVTFTTDTS without TSTAYMELRSLRSDDTAVYYCARWD linker
and FAYYVEAMDYWGQGTTVTVSSASTK <DOTAM- GPSVFPLAPSSKSTSGGTAALGCLV
VL>.fwdarw. Same KDYFPEPVTVSWNSGALTSGVHTFP Plasmid as
AVLQSSGLYSLSSVVTVPSSSLGTQ SeqID33, TYICNVNHKPSNTKVDKKVEPKSCD
lacking KTHTCPPCPAPEAAGGPSVFLFPPK linker and
PKDTLMISRTPEVTCVVVDVSHEDP <DOTAM> EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 30 P1AD8592
QVQLVQSGAEVKKPGASVKVSCKAS Heavy GYTFTEFGMNWVRQAPGQGLEWMGW chain
Knob IMTKTGEATYVEEFKGRVTFTTDTS <CEA>CH1A1A
TSTAYMELRSLRSDDTAVYYCARWD FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVFINAKTKPREEO YNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPRE PQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 31 Linker GGGGSGGGGSGGGGSGGGGS 32
P1AD8749 QVQLVQSGAEVKKPGASVKVSCKAS heavy GYTFTEFGMNWVRQAPGQGLEWMGW
chain knob INTKTGEATYVEEFKGRVTFTTDTS <CEA>CH1A1A
TSTAYMELRSLRSDDTAVYYCARWD <Dotam-VH>
FAYYVEAMDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSP VTLKES
GPVLVKPTETLTLTCTVSGFSLSTY SMSWIRQPPGKALEWLGFIGSRGDT
YYASWAKGRLTISKDTSKSQVVLTM TNMDPVDTATYYCARERDPYGGGAY PPHLWGRGTLVTVSS
33 P1AD8592 heavy QVQLVQSGAEVKKPGASVKVSCKAS chain hole
GYTFTEFGMNWVRQAPGQGLEWMGW <CEA>CH1A1A
INTKTGEATYVEEFKGRVTFTTDTS <Dotam-VL>
TSTAYMELRSLRSDDTAVYYCARWD FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG IQMTQ SPSSLSASVGDRVTITCQSSHSVYS
DNDLAWYQQKPGKAPKLLIYQASKL ASGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCLGGYDDESDTYGFGGG TKVEIK 34 P1AD8749 and
DIQMTQSPSSLSASVGDRVTITCKA P1AD8592 SAAVGTYVAWYQQKPGKAPKLLIYS light
chain ASYRKRGVPSRFSGSGSGTDFTLTI <CEA> CH1A1A
SSLQPEDFATYYCHQYYTYPLFTFG QGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQ 35 Heavy chain DYGVH CDR 1, <C825>
36 Heavy chain VIWSGGGTAYNTALIS CDR 2, <C825> 37 Heavy chain
RGSYPYNYFDA CDR 3, <C825> 38 Light chain GSSTGAVTASNYAN CDR
1, <C825> 39 Light chain GHNNRPP CDR 2, <C825> 40 Light
chain ALWYSDHWV CDR 3, <C825> 41 Heavy chain
HVKLQESGPGLVQPSQSLSLTCTVS variable GFSLTDYGVHWVRQSPGKGLEWLGV domain
IWSGGGTAYNTALISRLNIYRDNSK <C825> NQVFLEMNSLQAEDTAMYYCARRGS
YPYNYFDAWGQGTTVTVSS 42 Light chain QAVVIQESALTTPPGETVTLTCGSS
variable TGAVTASNYANWVQEKPDHLFTGLI domain,
GGHNNRPPGVPARFSGSLIGDKAAL <C825> TIAGTQTEDEAIYFCALWYSDHWVI
GGGTKLTVL 43 heavy chain DYYMN CDR1 <CEA> A5B7 44 heavy chain
FIGNKANAYTTEYSASVKG CDR2 <CEA> A5B7 45 heavy chain DRGLRFYFDY
CDR3 <CEA> A5B7 46 light chain RASSSVTYIH CDR1 <CEA>
A5B7 47 light chain ATSNLAS CDR2 <CEA> A5B7 48 light chain
QHWSSKPPT CDR3 <CEA> A5B7 49 heavy chain
EVQLLESGGGLVQPGGSLRLSCAAS variable domain GFTFTDYYMNWVRQAPGKGLEWLGF
<CEA> A5B7 IGNKANAYTTEYSASVKGRFTISRD
KSKNTLYLQMNSLRAEDTATYYCTR DRGLRFYFDYWGQGTTVTVSS 50 light chain
EIVLTQSPATLSLSPGERATLSCRA variable domain SSSVTYIHWYQQKPGQAPRSWIYAT
<CEA> A5B7 SNLASGIPARFSGSGSGTDFTLTIS
SLEPEDFAVYYCQHWSSKPPTFGQG TKLEIK 51 P1AE4956
EVQLLESGGGLVQPGGSLRLSCAAS heavy GFTFTDYYMNWVRQAPGKGLEWLGF chain
hole IGNKANAYTTEYSASVKGRFTISRD <CEA> A5B7
KSKNTLYLQMNSLRAEDTATYYCTR DRGLRFYFDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 52 P1AE4956 EVQLLESGGGLVQPGGSLRLSCAAS
heavy GFTFTDYYMNWVRQAPGKGLEWLGF chain knob
IGNKANAYTTEYSASVKGRFTISRD <CEA> A5B7
KSKNTLYLQMNSLRAEDTATYYCTR <Dolam-VH>
DRGLRFYFDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHKHYTQKSLSLSPG VTLKE
SGPVLVKPTETLTLTCTVSGFSLST YSMSWIRQPPGKALEWLGFIGSRGD
TYYASWAKGRLTISKDTSKSQVVLT MTNMDPVDTATYYCARERDPYGGGA
YPPHLWGRGTLVTVSS 53 Heavy chain EVQLLESGGGLVQPGGSLRLSCAAS
<CEA> of GFTFTDYYMNWVRQAPGKGLEWLGF P1AE4956
IGNKANAYTTEYSASVKGRFTISRD without KSKNTLYLQMNSLRAEDTATYYCTR linker
and DRGLRFYFDYWGQGTTVTVSSASTK DOTAM- GPSVFPLAPSSKSTSGGTAALGCLV
VH>.fwdarw. Same KDYFPEPVTVSWNSGALTSGVHTFP Plasmid
AVLQSSGLYSLSSVVTVPSSSLGTQ as SeqID 52, TYICNVNHKPSNTKVDKKVEPKSCD
lacking KTHTCPPCPAPEAAGGPSVFLFPPK linker and
PKDTLMISRTPEVTCVVVDVSHEDP <DOTAM> EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP 54 P1AE4956
EIVLTQSPATLSLSPGERATLSCRA light SSSVTYIHWYQQICPGQAPRSWIYA chain
<CEA> TSNLASGIPARFSGSGSGTDFTLTI A5B7
SSLEPEDFAVYYCQHWSSKPPTFGQ GTKLEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 55 P1AE4957
EVQLLESGGGLVQPGGSLRLSCAAS heavy chain GFTFTDYYMNWVRQAPGKGLEWLGF
knob <CEA> IGNKANAYTTEYSASVKGRFTISRD A5B7
KSKNTLYLQMNSLRAEDTATYYCTR DRGLRFYFDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 56 P1AE4957 EVQLLESGGGLVQPGGSLRLSCAAS
heavy chain GFTFTDYYMNWVRQAPGKGLEWLGF hole
IGNKANAYTTEYSASVKGRFTISRD <CEA> A5B7
KSKNTLYLQMNSLRAEDTATYYCTR <Dotam-VL>
DRGLRFYFDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLVSKLTVDKSRWOOGNV FSCSVMHEALHNHYTOKSLSLSPGG IQMTQS
PSSLSASVGDRVTITCQSSHSVYSD NDLAWYQQKPGKAPKLLIYQASKLA
SGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCLGGYDDESDTYGFGGGT KVEIK 57 Heavy
chain EVQLLESGGGLVQPGGSLRLSCAAS <CEA> of
GFTFTDYYMNWVRQAPGKGLEWLGF P1AE4957 IGNKANAYTTEYSASVKGRFTISRD
without KSKNTLYLQMNSLRAEDTATYYCTR linker DRGLRFYFDYWGQGTTVTVSSASTK
and DOTAM- GPSVFPLAPSSKSTSGGTAALGCLV VL>.fwdarw. Same
KDYFPEPVTVSWNSGALTSGVHTFP Plasmid as AVLQSSGLYSLSSVVTVPSSSLGTQ
SeqID 56, TYICNVNHKPSNTKVDKKVEPKSCD lacking
KTHTCPPCPAPEAAGGPSVFLFPPK linker PKDTLMISRTPEVTCVVVDVSHEDP and
<DOTAM> EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSP 58 P1AE4957 EIVLTQSPATLSLSPGERATLSCRA
light SSSVTYIHWYQQKPGQAPRSWIYAT chain <CEA>
SNLASGIPARFSGSGSGTDFTLTIS A5B7 SLEPEDFAVYYCQHWSSKPPTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
59 heavy chain GGTFSYYAIS CDR1 <CEA> 28A9 60 heavy chain
GILPAFGAANYAQKFQG CDR2 <CEA> 28A9 61 heavy chain LPPLPGAGLDY
CDR3 <CEA> 28A9
62 light chain RASQSISSWLA CDR1 <CEA> 28A9 63 light chain
DASSLES CDR2 <CEA> 28A9 64 light chain QQNTQYPMT CDR3
<CEA> 28A9 65 heavy chain QVQLVQSGAEVKKPGSSVKVSCKAS variable
GGTFSYYAISWVRQAPGQGLEWMGG domain ILPAFGAANYAQKFQGRVTITADK
<CEA> 28A9 STSTAYMELSSLRSEDTAVYYCARL PPLPGAGLDYWGQGTTVTVSS 66
light chain DIQMTQSPSTLSASVGDRVTITCRA variable
SQSISSWLAWYQQKPGKAPKLLIYD domain ASSLESGVPSRFSGSGSGTEFTLTI
<CEA> 28A9 SSLQPDDFATYYCQQNTQYPMTFGQ GTKVEIK 67 heavy chain
GFTFSKYAMA CDR1 <GPRC5D> 68 heavy chain SISTGGVNTYYADSVKG
CDR2 <GPRC5D> 69 heavy chain HTGDYFDY CDR3 <GPRC5D> 70
light chain RASQSVSISGINLMN CDR1 <GPRC5D> 71 light chain
HASILAS CDR2 <GPRC5D> 72 light chain QQTRESPLT CDR3
<GPRC5D> 73 Heavy chain EVQLLESGGGLVQPGGSLRLSCAAS variable
GFTFSKYAMAWVRQAPGKGLEWVAS domain ISTGGVNTYYADSVKGRFTISRDNS
<GPRC5D> KNTLYLQMNSLRAEDTAVYYCATHT GDYFDYWGQGTMVTVSS 74 Light
chain EIVLTQSPGTLSLSPGERATLSCRA variable SQSVSISGINLMNWYQQKPGQQPKL
domain LIYHASILASGIPDRFSGSGSGTDF <GPRC5D>
TLTISRLEPEDFAVYYCQQTRESPL TFGQGTRLEIK 75 heavy chain GFTFSSYAMS
CDR1 <FAP> 4B9 76 heavy chain AIIGSGASTYYADSVKG CDR2
<FAP> 4B9 77 heavy chain GWFGGFNY CDR3 <FAP> 4B9 78
light chain RASQSVTSSYLA CDR1 <FAP> 4B9 79 light chain
VGSRRAT CDR2 <FAP> 4B9 80 light chain QQGIMLPPT CDR3
<FAP> 4B9 81 Heavy chain EVQLLESGGGLVQPGGSLRLSCAAS variable
GFTFSSYAMSWVRQAPGKGLEWVSA domain IIGSGASTYYADSVKGRFTISRDNS
<FAP> 4B9 KNTLYLQMNSLRAEDTAVYYCAKGW FGGFNYWGQGTLVTVSS 82
Light chain EIVLTQSPGTLSLSPGERATLSCRA variable
SQSVTSSYLAWYQQKPGQAPRLLIN domain VGSRRATGIPDRFSGSGSGTDFTLT
<FAP> 4B9 ISRLEPEDFAVYYCQQGIMLPPTFG QGTKVEIK 83 P1AF0709
QVQLVQSGAEVKKPGSSVKVSCKAS HCknob <CEA>
GFNIKDTYMHWVRQAPGQGLEWMGR T84.66 IDPANGNSKYVPKFQGRVTITADTS
(D1AE4688) TSTAYMELSSLRSEDTAVYYCAPFG YYVSDYAMAYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALGAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWC
LVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLS LSPG 84 P1AF0709
QVQLVQSGAEVKKPGSSVKVSCKAS HChole <CEA>
GFNIKDTYMHWVRQAPGQGLEWMGR T84.66 Dotam- IDPANGNSKYVPKFQGRVTITADTS
VL TSTAYMELSSLRSEDTAVYYCAPFG (D1AA4920) YYVSDYAMAYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGSIQMTQ
SPSSLSASVGDRVTITCQSSHSVYS DNDLAWYQQKPGKAPKLLIYQASKL
ASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCLGGYDDESDTYGFGGG TKVEflC 85
P1AF0709 QVQLVQSGAEVKKPGSSVKVSCKAS HChole <CEA>
GFNIKDTYMHWVRQAPGQGLEWMGR T84.66 IDPANGNSKYVPKFQGRVTITADTS without
TSTAYMELSSLRSEDTAVYYCAPFG linker and YYVSDYAMAYWGQGTLVTVSSASTK
DOTAM GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKXVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 86 P1AF0298 QVQLVQSGAEVKKPGSSVKVSCKAS
HCHole <CEA> GFNTKDTYMHWVRQAPGQGLEWMGR T84.66
IDPANGNSKYVPKFQGRVTITADTS (D1AE4687) TSTAYMELSSLRSEDTAVYYCAPFG
YYVSDYAMAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 87 P1AF0298
QVQLVQSGAEVKKPGSSVKVSCKAS HCknob <CEA>
GFNIKDTYMHWVRQAPGQGLEWMGR T84.66 IDPANGNSKYVPKFQGRVTITADTS Dotam-
TSTAYMELSSLRSEDTAVYYCAPFG VH-AST YYVSDYAMAYWGQGTLVTVSSASTK
(D1AE3668) GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGSVTLKE
SGPVLVKPTETLTLTCTVSGFSLST YSMSWIRQPPGKALEWLGFIGSRGD
TYYASWAKGRLTISKDTSKSQVVLT MTNMDPVDTATYYCARERDPYGGGA
YPPHLWGRGTLVTVSSAST 88 P1AF0298 QVQLVQSGAEVKKPGSSVKVSCKAS HCknob
<CEA> GFNIKDTYMHWVRQAPGQGLEWMGR T84.66
IDPANGNSKYVPKFQGRVTITADTS without TSTAYMELSSLRSEDTAVYYCAPFG linker
and YYVSDYAMAYWGQGTLVTVSSASTK DOTAM GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 89 P1AF0709 and
EIVLTQSPATLSLSPGERATLSCRA P1AF0298 GESVDIFGVGFLHWYQQKPGQAPRL light
LIYRASNRATGIPARFSGSGSGTDF chain TLTISSLEPEDFAVYYCQQTNEDPY
(D1AA4120) TFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC 90 P1AF0710 QVQLVQSGAEVKKPGSSVKVSCKAS HCknob
<CEA> GGTFSYYAISWVRQAPGQGLEWMGG 28A9
ILPAFGAANYAQKFQGRVTITADKS (D1AE4690) TSTAYMELSSLRSEDTAVYYCARLP
PLPGAGLDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK
DYPPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKV SNKALGAPIEKTISKAKGQPREPQV
YTLPPCRDELTKNQVSLWCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG
91 P1AF0710 QVQLVQSGAEVKKPGSSVKVSCKAS HChole <CEA>
GGTFSYYAISWVRQAPGQGLEWMGG 28A9 Dotam- ILPAFGAANYAQKFQGRVTITADKS VL
TSTAYMELSSLRSEDTAVYYCARLP (D1AC3172) PLPGAGLDYWGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCK
VSNKALGAPIEKTISKAKGQPREPQ VCTLPPSRDELTKNQVSLSCAVKGF
YPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLVSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGG GGGSGGGGSGGGGSGGGGSIQMTQS
PSSLSASVGDRVTITCQSSHSVYSD NDLAWYQQKPGKAPKLLIYQASKLA
SGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCLGGYDDESDTYGFGGGT KVEEK 92
P1AF0710 QVQLVQSGAEVKKPGSSVKVSCKAS HChole <CEA>
GGTFSYYAISWVRQAPGQGLEWMGG 28A9 without ILPAFGAANYAQKFQGRVTLTADKS
linker or TSTAYMELSSLRSEDTAVYYCARLP DOTAM PLPGAGLDYWGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCK
VSNKALGAPIEKTISKAKGQPREPQ VCTLPPSRDELTKNQVSLSCAVKGF
YPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLVSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPG 93 P1AF0711 QVQLVQSGAEVKKPGSSVKVSCKAS
HChole <CEA> GGTFSYYAISWVRQAPGQGLEWMGG 28A9
ILPAFGAANYAQKFQGRVTITADKS (D1AE4689) TSTAYMELSSLRSEDTAVYYCARLP
PLPGAGLDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQ
VCTLPPSRDELTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 94 P1AF0711
QVQLVQSGAEVKKPGSSVKVSCKAS HCknob <CEA>
GGTFSYYAISWVRQAPGQGLEWMGG 28A9 Dotam- ILPAFGAANYAQKFQGRVTITADKS
VH-AST TSTAYMELSSLRSEDTAVYYCARLP (D1AE3671)
PLPGAGLDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGG
GGGSGGGGSGGGGSGGGGSVTLKES GPVLVKPTETLTLTCTVSGFSLSTY
SMSWIRQPPGKALEWLGFIGSRGDT YYASWAKGRLTISKDTSKSQVVLTM
TNMDPVDTATYYCARERDPYGGGAY PPHLWGRGTLVTVSSAST 95 P1AF0711
QVQLVQSGAEVKKPGSSVKVSCKAS HCknob <CEA>
GGTFSYYAISWVRQAPGQGLEWMGG 28A9 without ILPAFGAANYAQKFQGRVTITADKS
linker and TSTAYMELSSLRSEDTAVYYCARLP DOTAM
PLPGAGLDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCK VSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 96 P1AF0710 and
DIQMTQSPSTLSASVGDRVTITCRA P1AF0711 SQSISSWLAWYQQKPGKAPKLLIYD light
ASSLESGVPSRFSGSGSGTEFTLTI chain SSLQPDDFATYYCQQNTQYPMTFGQ
(D1AA2299) GTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
97 P1AF0712 QVQLVQSGAEVKKPGASVKVSCKAS HCknob <CEA>
GYTFTEFGMNWVRQAPGQGLEWMGW CH1Al A INTKTGEATYVEEFKGRVTFTT (D1AC4023)
DTSTSTAYMELRSLRSDDTAVYYCA RWDFAYYVEAMDYWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVE
PKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVS
LWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 98 P1AF0712
QVQLVQSGAEVKKPGASVKVSCKAS HChole <CEA>
GYTFTEFGMNWVRQAPGQGLEWMGW CH1A1A INTKTGEATYVEEFKGRVTFTTDTS DOTA-VL
TSTAYMELRSLRSDDTAVYYCARWD (D1AE4684) FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGSQAVVI
QESALTTPPGETVTLTCGSSTGAVT ASNYANWVQEKPDHLFTGLIGGHNN
RPPGVPARFSGSLIGDKAALTIAGT QTEDEAIYFCALWYSDHWVIGGGTK LTVL 99
P1AF0712 QVQLVQSGAEVKKPGASVKVSCKAS HChole <CEA>
GYTFTEFGMNWVRQAPGQGLEWMGW without INTKTGEATYVEEFKGRVTFTT linker or
DTSTSTAYMELRSLRSDDTAVYYCA DOTA RWDFAYYVEAMDYWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVE
PKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAK GQPREPQVCTLPPSRDELTKNQVS
LSCAVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLVSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 100 P1AF0713
QVQLVQSGAEVKKPGASVKVSCKAS HCHole <CEA>
GYTFTEFGMNWVRQAPGQGLEWMGW CH1A1A INTKTGEATYVEEFKGRVTFTTDTS
(D1AC4022) TSTAYMELRSLRSDDTAVYYCARWD FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 101 P1AF0713 QVQLVQSGAEVKKPGASVKVSCKAS
HCknob <CEA> GYTFTEFGMNWVRQAPGQGLEWMGW CH1A1A
INTKTGEATYVEEFKGRVTFTTDTS DOTA-VH-AST TSTAYMELRSLRSDDTAVYYCARWD
(D1AE3670) FAYYVEAMDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG
GGGGSGGGGSGGGGSGGGGSHVKLQ ESGPGLVQPSQSLSLTCTVSGFSLT
DYGVHWVRQSPGKGLEWLGVIWSGG GTAYNTALISRLNIYRDNSKNQVFL
EMNSLQAEDTAMYYCARRGSYPYNY FDAWGQGTTVTVSSAST 102 P1AF0713
QVQLVQSGAEVKKPGASVKVSCKAS HCknob <CEA>
GYTFTEFGMNWVRQAPGQGLEWMGW CH1A1A INTKTGEATYVEEFKGRVTFTTDTS without
TSTAYMELRSLRSDDTAVYYCARWD linker FAYYVEAMDYWGQGTTVTVSSASTK and DOTA
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG 103 P1AF0712 DIQMTQSPSSLSASVGDRVTITCKA
and SAAVGTYVAWYQQKPGKAPKLLIYS P1AF0713 ASYRKRGVPSRFSGSGSGTDFTLTI
light SSLQPEDFATYYCHQYYTYPLFTFG chain QGTKLEIKRTVAAPSVFIFPPSDEQ
(D1AA3384) LKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 104 P1AF8284 and
EVQLLESGGGLVQPGGSLRLSCAAS P1AF8285 GFTFSKYAMAWVRQAPGKGLEWVAS HCknob
ISTGGVNTYYADSVKGRFTISRDNS <GPRC5D> KNTLYLQMNSLRAEDTAVYYCATHT
(D1AF6517) GDYFDYWGQGTMVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYT
LPPCRDELTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALIINIIYTQKSLSLSPGK 105 P1AF8284
DKTHTCPPCPAPEAAGGPSVFLFPP HChole Dotam- KPKDTLMISRTPEVTCVVVDVSHED
VL PEVKFNWYVDGVEVHNAKTKPREEQ (D1AG3592) YNSTYRWSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG KGGGGSGGGGSGGGGSGGGGSSIQM
TQSPSSLSASVGDRVTITCQSSHSV YSDNDLAWYQQKPGKAPKLLIYQAS
KLASGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCLGGYDDESDTYGFG GGTKVEEK 106
P1AF8285 DKTHTCPPCPAPEAAGGPSVFLFPP Hhole Dotam-
KPKDTLMISRTPEVTCVVVDVSHED VHA PEVKFNWYVDGVEVHNAKTKPREEQ (D1AG3591)
YNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALGAPIEKTISKAKGQPRE
PQVCTLPPSRDELTKNQVSLSCAVK GFYPSDLAVEWESNGQPENNYKTTP
PVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSP
GKGGGGSGGGGSGGGGSGGGGSVTL KESGPVLVKPTETLTLTCTVSGFSL
STYSMSWIRQPPGKALEWLGFIGSR GDTYYASWAKGRLTISKDTSKSQVV
LTMTNMDPVDTATYYCARERDPYGG GAYPPHLWGRGTLVTVSSA 107 P1AF8284
EIVLTQSPGTLSLSPGERATLSCRA and SQSVSISGINLMNWYQQKPGQQPKL P1AF8285
LIYHASILASGIPDRFSGSGSGTDF light TLTISRLEPEDFAVYYCQQTRESPL chain
TFGQGTRLEIKRTVAAPSVFIFPPS (D1AF6469) DEQLKSGTASVVCLLNNFYPREAIC
VQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC 108 P1AF8286 EVQLLESGGGLVQPGGSLRLSCAAS and
GFTFSSYAMSWVRQAPGKGLEWVSA P1AF8287 IIGSGASTYYADSVKGRFTISRDNS Hcknob
KNTLYLQMNSLRAEDTAVYYCAKGW <FAP> FGGFNYWGQGTLVTVSSASTKGPSV 4B9
FPLAPSSKSTSGGTAALGCLVKDYF (D1AF6515) PEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNK
ALGAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 109 P1AF8286 DKTIITCPPCPAREAAGGPSVFLFP HChole
Dotam- PKPKDTLMISRTPEVTCVVVDVSII VL NDPEVKPNWYVDGVEVHNAXTKPRE
(D1AG3592) EQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALGAPIEKTISKAKGQP
REPQVCTLPPSRDELTKNQVSLSCA VKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSL
SPGKGGGGSGGGGSGGGGSGGGGSS IQMTQSPSSLSASVGDRVTITCQSS
HSVYSDNDLAWYQQKPGKAPKLLIY QASKLASGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCLGGYDDESDTY GFGGGTKVEIK 110 P1AF8287
DKTHTCPPCPAPEAAGGPSVFLFPP Hchole KPKDTLMISRTPEVTCVVVDVSHED Dotam-
PEVKFNWTVDGVEVHNAKTKPREEQ VHA YNSTYRVVSVLTVLHQDWLNGKEYK (D1AG3591)
CKVSNKALGAPIEKTISKAKGQPRE PQVCTLPPSRDE LTKNQVSLSCAVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKGGGGSGGGGS GGGGSGGGGSVTLKESGPVLVKPTE
TLTLTCTVSGFSLSTYSMSWIRQPP GKALEWLGFIGSRGDTYYASWAKGR
LTISKDTSKSQVVLTMTNMDPVDTA TYYCARERDPYGGGAYPPHLWGRGT LVTVSSA 111
P1AF8286 EIVLTQSPGTLSLSPGERATLSCRA and SQSVTSSYLAWYQQKPGQAPRLLIN
P1AF8287 VGSRRATGIPDRFSGSGSGTDFTLT light ISRLEPEDFAVYYCQQGIMLPPTFG
chain QGTKVEIKRTVAAPSVFIFPPSDEQ (D1AB9974)
LKSGTASVVCLLNNFYPREAKVQWT CVDN ALQSGNSQESVTEODSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC 112 P1AF7782 and
QVQLVQSGAEVKKPGASVKVSCKAS P1AF7784 GYTFTEFGMNWVRQAPGQGLEWMGW HCknob
<CEA> INTKTGEATYVEEFKGRVTFTTDTS CH1A1A
TSTAYMELRSLRSDDTAVYYCARWD (D1AD3419) FAYYVEAMDYWGQGTTVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALGAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKG
FYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG K 113 P1AF7782 SIQMTQSPSSLSASVGDRVTITCQS
Hchole SHSVYSDNDLAWTQQKPGKAPKLLI Dotam- YQASKLASGVPSRFSGSGSGTDFTL
VL TISSLQPEDFATYYCLGGYDDESDT (D1AG2237) YGFGGGTKVEIKGGGGSGGGGSGGG
GSGGSGGDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVCTLPPSRDELTKNQV
SLSCAVKGFYPSDLAVEWESNGQPE NNYKTTPPVLDSDGSFFLVSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 114 P1AF7784
GVTLKESGPVLVKPTETLTLTCTVS Hchole GFSLSTYSMSWIRQPPGKALEWLGF Dotam-
IGSRGDTYYASWAKGRLTISKDTSK VH S (D1AG2236) QVVLTMTNMDPVDTATYYCARERDP
YGGGAYPPHLWGRGTLVTVSSGGGG SGGGGSGGGGSGGSGGDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVS
NKALGAPIEKTISKAKGQPREPQVC TLPPSRDELTKNQVSLSCAVKGFYP
SDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFS
CSVMHEALITNITYTQKSLSLSPGK 115 P1AF7782 DIQMTQSPSSLSASVGDRVTITCKA
and SAAVGTYVAWYQQKPGKAPKLLIYS P1AF7784 ASYRKRGVPSRFSGSGSGTDFTLTI
light SSLQPEDFATYYCHQYYTYPLFTFG chain QGTKLEIKRTVAAPSVFIFPPSDEQ
(D1AD3421) LKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 156 heavy chain DSYMH
CDR1 <CEA> MFE23 157 heavy chain WrDPENGDTEYAPKFQG CDR2
<CEA> MFE23 158 heavy chain WIDPENGGTNYAQKFQG CDR2
<CEA> MFE23-H26 159 heavy chain GTPTGPYYFDY CDR3 <CEA>
MFE23 160 light chain SASSSVSYMH CDR1 <CEA> MFE23 161 light
chain RASSSVSYMH CDR1 <CEA> MFE23-L24, L25 162 light chain
RASQSISSYM CDR1<CEA> MFE23-L26 163 light chain STSNLAS
CDR2<CEA> MFE23 164 Light chain YTSNLAS CDR2<CEA>
MFE23-L26 165 Light chain STSSLQS CDR2<CEA> MFE23-L29 166
light chain QQRSSYPLT CDR3<CEA> MFE23 167 Heavy chain
QVKLQQSGAELVRSGTSVKLSCTAS variable GFNTKDSYMHWLRQGPEQGLEWIGW domain
IDPENGDTEYAPKFQGKATFTTDTS <CEA> MFE23
SNTAYLQLSSLTSEDTAVYYCNEGT PTGPYYFDYWGQGTTVTVSS 168 Light chain
ENVLTQSPAIMSASPGEKVTITCSA variable SSSVSYMHWFQQKPGTSPKLWTYST domain
SNLASGVPARFSGSGSGTSYSLTIS <CEA> MFE23
RMEAEDAATYYCQQRSSYPLTFGAG TKLELK 169 MFE-H24
QVQLVQSGAEVKKPGASVKVSCKAS GFNIKDSYMHWVRQAPGQGLEWMGW
IDPENGDTEYAPKFQGRVTMTTDTS ISTAYMELSRLRSDDTAVYYCNEGT
PTGPYYFDYWGQGTLVTVSS 170 MFE-H25 QVQLVQSGAEVKKPGASVKVSCKAS
GYTFKDSYMHWVRQAPGQGLEWMGW IDPENGDTEYAPKFQGRVTMTTDTS
ISTAYMELSRLRSDDTAVYYCNEGT PTGPYYFDYWGQGTLVTVSS 171 MFE-H26
QVQLVQSGAEVKKPGASVKVSCKAS GFNIKDSYMHWVRQAPGQGLEWMGW
IDPENGGTNYAQKFQGRVTMTTDTS ISTAYMELSRLRSDDTAVYYCNEGT
PTGPYYFDYWGQGTLVTVSS 172 MFE-H27 QVQLVQSGAEVKKPGASVKVSCKAS
GFNIKDSYMHWVRQAPGQGLEWMGW IDPENGDTEYAPKFQGRVTMTTDTS
ISTAYMELSRLRSDDTAVYYCARGT PTGPYYFDYWGQGTLVTVSS 173 MFE-H28
QVQLVQSGAEVKKPGASVKVSCKAS GFNIKDSYMHWVRQAPGQGLEWMGW
IDPENGDTEYAPKFQGRVTMTRDTS ISTAYMELSRLRSDDTAVYYCNEGT
PTGPYYFDYWGQGTLVTVSS 174 MFE-H29 QVQLVQSGAEVKKPGSSVKVSCKAS
GFNIKDSYMHWVRQAPGQGLEWMGW IDPENGDTEYAPKFQGRVTNTDEST
STAYMELSSLRSEDTAVYYCNEGTP TGPYYFDYWGQGTLVTVSS 175 MFE-L24
DIQMTQSPSSLSASVGDRVTITCRA SSSVSYMHWYQQKPGKAPKLLIYST
SNLASGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQRSSYPLTFGGG TKLEIK 176
MFE-L25 EIQMTQSPSSLSASVGDRVTITCRA SSSVSYMHWYQQKPGKAPKLLIYST
SNLASGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQRSSYPLTFGGG TKLEIK 177
MFE-L26 EIQMTQSPSSLSASVGDRVTITCRA SQSISSYMHWYQQKPGKAPKLLIYS
TSNLASGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQRSSYPLTFGG GTKLEIK 178
MFE-L27 EIQMTQSPSSLSASVGDRVTITCRA SSSVPYMHWYQQKPGKAPKLLIYST
SNLASGVPSRFSGSGSGTDFTLTIS SVQPEDFATYYCQQRSSYPLTFGGG TKLEIK 179
MFE-L28 EIQMTQSPSSLSASVGDRVTITCRA SSSVPYMHWLQQKPGKAPKLLIYST
SNLASGVPSRFSGSGSGTDFTLTIS SVQPEDFATYYCQQRSSYPLTFGGG TKLEIK 180
MFE-L29 EIQMTQSPSSLSASVGDRVTITCRA SSSVPYMHWLQQKPGKAPKLLIYST
SSLQSGVPSRFSGSGSGTDFTLTIS SVQPEDFATYYCQQRSSYPLTFGGG TKLEIK
IV. Examples
[0712] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Glossary of Abbreviations
[0713] ADA Anti-drug antibody AST Alanine, serine, threonine BsAb
Bispecific antibody CA Clearing agent CEA Carcinoembryonic antigen
DOTAM
1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane
ID Injected dose ELISA Enzyme-linked immunosorbent assay FAP
Fibroblast activation protein GPRC5D G-protein coupled receptor
family C group 5 member D
IV Intravenous
[0714] MW Molecular weight PBS Phosphate-buffered saline p.i. Post
injection
PK Pharmacokinetic
[0715] PRIT Pretargeted radioimmunotherapy
RIT Radioimmunotherapy
[0716] RT Room temperature
SC Subcutaneous
[0717] SCID Severe combined immunodeficiency SD Standard deviation
SOPF Specific and opportunistic pathogen-free TA Target antigen TGI
Tumor growth inhibition TR Tumor regression
Example 1: Generation of Rabbit DOTAM Binding Antibodies
Example 1a: Immunization of Rabbits
[0718] A 1:1 mix of the 2 enantiomeric Pb-DOTAM-alkyl-PEG.sub.4-KLH
fractions (MS2-DOTAM KLH Fraction 1 and MS2-DOTAM KLH Fraction 2)
was used for the immunization of New Zealand White rabbits or
transgenic rabbits comprising a human immunoglobulin locus as
reported in WO 2000/46251, WO 2002/12437, WO 2005/007696, WO
2006/047367, US 2007/0033661, and WO 2008/027986. Each rabbit was
immunized with 500 ug of the immunogen mix, emulsified with
complete Freund's adjuvant, at day 0 by intradermal application and
500 ug each at days 7, 14, 28, 56 by alternating intramuscular and
subcutaneous applications. Thereafter, rabbits received monthly
subcutaneous immunizations of 500 ug, and small samples of blood
were taken 7 days after immunization for the determination of serum
titers. A larger blood sample (10% of estimated total blood volume)
was taken during the third and during the ninth month of
immunization (at 5-7 days after immunization), and peripheral
mononuclear cells were isolated, which were used as a source of
antigen-specific B cells in the B cell cloning process.
Determination of Serum Titers (ELISA)
[0719] Each of the 2 enantiomeric Pb-DOTAM fractions (PJRD05.133F1
or PJRD05.133F2) was immobilized on a 96-well NUNC Maxisorp plate
at 1 ug/ml, 100 ul/well, in PBS, followed by: blocking of the plate
with 2% Crotein C in PBS, 200 ul/well; application of serial
dilutions of antisera, in duplicates, in 0.5% Crotein C in PBS, 100
ul/well; detection with HRP-conjugated donkey anti-rabbit IgG
antibody (Jackson Immunoresearch/Dianova 711-036-152; 1/16 000) and
streptavidin-HRP; each diluted in 0.5% Crotein C in PBS, 100
ul/well. For all steps, plates were incubated for 1 h at 37.degree.
C. Between all steps, plates were washed 3 times with 0.05% Tween
20 in PBS. Signal was developed by addition of BM Blue POD
Substrate soluble (Roche), 100 ul/well; and stopped by addition of
1 M HC1, 100 ul/well. Absorbance was read out at 450 nm, against
690 nm as reference. Titer was defined as dilution of antisera
resulting in half-maximal signal.
Example 1B: B-Cell Cloning from Rabbits
Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMCs)
[0720] Blood samples were taken of immunized rabbits. EDTA
containing whole blood was diluted twofold with 1.times.PBS (PAA,
Pasching, Austria) before density centrifugation using lympholyte
mammal (Cedarlane Laboratories, Burlington, Ontario, Canada)
according to the specifications of the manufacturer. The PBMCs were
washed twice with 1.times.PBS.
EL-4 B5 Medium
[0721] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented
with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM Glutamin, 1%
penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM
sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and
0.05 mM b-mercaptoethanole (Gibco, Paisley, Scotland) was used.
Coating of Plates
[0722] Sterile cell culture 6-well plates were coated with 2
.mu.g/ml KLH in carbonate buffer (0,1 M sodium bicarbonate, 34 mM
Disodiumhydrogencarbonate, pH 9,55) overnight at 4.degree. C.
Plates were washed in sterile PBS three times before use. Sterile
streptavidin coated 6-well plates (Microcoat, Bernried, Germany)
were coated with a 1+1 enantiomer mixture of biotinylated
TCMC-Pb-dPEC3-Biotin Isomer A (1 .mu.g/ml) and B (1 .mu.g/ml) in
PBS for 3 h at room temperature. Prior to the panning step these
6-well plates were washed three times with sterile PBS.
Depletion of Macrophages/Monocytes
[0723] The PBMCs were seeded on sterile KLH-coated 6-well-plates to
deplete macrophages and monocytes through unspecific adhesion and
to remove cells binding to KLH. Each well was filled at maximum
with 4 ml medium and up to 6.times.10e6 PBMCs from the immunized
rabbit and were allowed to bind for 1 h at 37.degree. C. and 5%
CO2. The cells in the supernatant (peripheral blood lymphocytes
(PBLs)) were used for the antigen panning step.
Enrichment of B Cells on the Pb-Containing TCMC Enantiomer
[0724] 6-well plates coated with the enantiomer mixture of
TCMC-Pb-dPEC3-Biotin Isomer A and B were seeded with up to
6.times.10e6 PBLs per 4 ml medium and allowed to bind for 1 h at
37.degree. C. and 5% CO2. Non-adherent cells were removed by
carefully washing the wells 1-3 times with 1.times.PBS. The
remaining sticky cells were detached by trypsin for 10 min at
37.degree. C. and 5% CO2. Trypsination was stopped with EL-4 B5
medium. The cells were kept on ice until the immune fluorescence
staining.
Immune Fluorescence Staining and Flow Cytometry
[0725] The anti-IgG FITC (AbD Serotec, Dusseldorf, Germany) was
used for single cell sorting. For surface staining, cells from the
depletion and enrichment step were incubated with the anti-IgG FITC
antibody in PBS and incubated for 45 min in the dark at 4.degree.
C. After staining the PBMCs were washed two times with ice cold
PBS. Finally the PBMCs were resuspended in ice cold PBS and
immediately subjected to the FACS analyses. Propidium iodide in a
concentration of 5 .mu.g/ml (BD Pharmingen, San Diego, Calif., USA)
was added prior to the FACS analyses to discriminate between dead
and live cells.
[0726] A Becton Dickinson FACSAria equipped with a computer and the
FACSDiva software (BD Biosciences, USA) were used for single cell
sort.
B-Cell Cultivation
[0727] The cultivation of the rabbit B cells was prepared by a
method described by Lightwood et al (J Immunol Methods, 2006, 316:
133-143). Briefly, single sorted rabbit B cells were incubated in
96-well plates with 200 .mu.l/well EL-4 B5 medium containing
Pansorbin Cells (1:100000) (Calbiochem (Merck), Darmstadt,
Deutschland), 5% rabbit thymocyte supernatant (MicroCoat, Bernried,
Germany) and gamma-irradiated murine EL-4 B5 thymoma cells
(5.times.10e5 cells/well) for 7 days at 37.degree. C. in the
incubator. The supernatants of the B-cell cultivation were removed
for screening and the remaining cells were harvested immediately
and were frozen at -80.degree. C. in 100 .mu.l RLT buffer (Qiagen,
Hilden, Germany).
Example 1C: Expression of Rabbit Antibody
PCR Amplification of V-Domains
[0728] Total RNA was prepared from B cells lysate (resuspended in
RLT buffer--Qiagen--Cat. N.sup.o 79216) using the NucleoSpin 8/96
RNA kit (Macherey&Nagel; 740709.4, 740698) according to
manufacturer's protocol. RNA was eluted with 60 .mu.l RNase free
water. 6 .mu.l of RNA was used to generate cDNA by reverse
transcriptase reaction using the Superscript III First-Strand
Synthesis SuperMix (Invitrogen 18080-400) and an oligo dT-primer
according to the manufacturers' instructions. All steps were
performed on a Hamilton ML Star System. 4 .mu.l of cDNA were used
to amplify the immunoglobulin heavy and light chain variable
regions (VH and VL) with the AccuPrime Supermix (Invitrogen
12344-040) in a final volume of 50 .mu.l using the primers rbHC.up
and rbHC.do for the heavy chain and rbLC.up and rbLC.do for the
light chain (Table below). All forward primers were specific for
the signal peptide (of respectively VH and VL) whereas the reverse
primers were specific for the constant regions (of respectively VH
and VL). The PCR conditions for the RbVH+RbVL were as follows: Hot
start at 94.degree. C. for 5 min; 35 cycles of 20 s at 94.degree.
C., 20 s at 70.degree. C., 45 s at 68.degree. C., and a final
extension at 68.degree. C. for 7 min.
Primer Sequences
TABLE-US-00012 [0729] rbHC.up AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC
(SEQ ID NO.: 141) rbHCf.do CCATTGGTGAGGGTGCCCGAG (SEQ ID NO.: 142)
rbLC.up AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC (SEQ ID NO.: 143)
rbLC.do CAGAGTRCTGCTGAGGTTGTAGGTAC (SEQ ID NO.: 144)
[0730] 8 .mu.l of 50 .mu.l PCR solution were loaded on a 48 E-Gel
2% (Invitrogen G8008-02). Positive PCR reactions were cleaned using
the NucleoSpin Extract II kit (Macherey&Nagel; 740609250)
according to manufacturer's protocol and eluted in 50 .mu.l elution
buffer. All cleaning steps were performed on a Hamilton ML Starlet
System.
Recombinant Expression of Rabbit Monoclonal Bivalent Antibodies
[0731] For recombinant expression of rabbit monoclonal bivalent
antibodies, PCR-products coding for VH or VL were cloned as cDNA
into expression vectors by the overhang cloning method (R S Haun et
al., Biotechniques (1992) 13, 515-518; MZ Li et al., Nature Methods
(2007) 4, 251-256). The expression vectors contained an expression
cassette consisting of a 5' CMV promoter including intron A, and a
3' BGH poly adenylation sequence. In addition to the expression
cassette, the plasmids contained a pUC18-derived origin of
replication and a beta-lactamase gene conferring ampicillin
resistance for plasmid amplification in E. coli. Three variants of
the basic plasmid were used: one plasmid containing the rabbit IgG
constant region designed to accept the VH regions while two
additional plasmids containing rabbit or human kappa LC constant
region to accept the VL regions. Linearized expression plasmids
coding for the kappa or gamma constant region and VL/VH inserts
were amplified by PCR using overlapping primers. Purified PCR
products were incubated with T4 DNA-polymerase which generated
single-strand overhangs. The reaction was stopped by dCTP addition.
In the next step, plasmid and insert were combined and incubated
with recA which induced site specific recombination. The recombined
plasmids were transformed into E. coli. The next day the grown
colonies were picked and tested for correct recombined plasmid by
plasmid preparation, restriction analysis and DNA-sequencing. For
antibody expression, the isolated HC and LC plasmids were
transiently co-transfected into 2 ml (96 well plate) of FreeStyle
HEK293-F cells (Invitrogen R790-07) by using 239-Free Transfection
Reagent (Novagen) following procedure suggested by Reagent
supplier. The supernatants were harvested after 1 week and
delivered for purification.
Example 1D: Selection of Rabbit Monoclonal Antibodies
[0732] A SET (solution equilibration titration) assay was carried
out as described below.
SET Assay
Materials:
[0733] 1. DOTAM-Biotin-Isomer Mix: [0734] Mixture of the following
components, conc.=20 ng/ml [0735]
Pb-Dotam-Bn-biotin/TCMC-Pb-dPEG3-Biotin, isomer A [0736]
Pb-Dotam-Bn-biotin/TCMC-Pb-dPEG3-Biotin, isomer B [0737]
Pb-Dotam-alkyl-biotin isomer A [0738] Pb-Dotam-alkyl-biotin isomer
B [0739] 2. PBS: DPBS, PAN, P04-36500 [0740] 3. BSA: Roche,
10735086001 [0741] 4. Tween 20: Polysorbat 20 (usb, #20605, 500 ml)
[0742] 5. PBST: 10.times., Roche, #11666789001/0,1% Tween 20 [0743]
6. OSEP: PBS (10.times., Roche, #11666789001)/0,5% BSA (Bovine
Serum Albumin Fraction V, fatty acid free, Roche,
#10735086001)/0,05% Tween 20
[0744] Preparation of assay-plate: 384-well streptavidin plates
(Nunc, Microcoat #11974998001) were incubated overnight at
4.degree. C. with 25 .mu.l/well of a DOTAM-Biotin-Isomer Mix in
PBS-buffer at a concentration of 20 ng/ml.
[0745] Equilibration of anti-DOTAM antibody samples with free
DOTAM-metal chelates (Pb, Bi, Ca, Cu, Zn, Mg, Fe): 0.01 nM-1 nM of
antibody were titrated with the relevant DOTAM-metal chelates in
1:3, 1:2 or 1:1.7 dilution steps starting at a concentration of
2500 nM, 500 nM or 100 nM of DOTAM-metal chelate. The samples were
incubated at 4.degree. C. overnight in sealed REMP Storage
polypropylene microplates (Brooks).
[0746] After overnight incubation, streptavidin plates were washed
3.times. with 90 .mu.l PBST per well. 15 .mu.l of each sample from
the equilibration plate were transferred to the assay plate and
incubated for 15 min at RT, followed by 3.times.90 .mu.l washing
steps with PBST buffer. Detection was carried out by adding 25
.mu.l of a goat anti-human IgG antibody-POD conjugate (Jackson,
109-036-088, 1:4000 in OSEP), followed by 6.times.90 .mu.l washing
steps with PBST buffer. 25 .mu.l of TMB substrate (Roche
Diagnostics GmbH, Cat. No.: 11835033001) were added to each well.
Measurement took place at 370/492 nm on a Safire2 reader
(Tecan).
[0747] The table below shows the properties of various monoclonal
bivalent rabbit antibodies as determined using this assay.
PRIT-0128 was selected as the lead candidate as it has comparable
binding to chelated Pb and Bi, reduced binding to other chelated
metals, and high affinity (<100 pM).
Binding of Monoclonal Bivalent Rabbit Antibodies to Chelated
Metals
TABLE-US-00013 [0748] KD [SET-Titration] Name Species Pb Bi Ca Zn
PRIT-0135 WTRa 0.000 0.000 0.001 0.285 PRIT-0129 WTRa 0.001 0.024
0.013 9.251 PRIT-0128 WTRa 0.002 0.003 0.003 8.152 PRIT-0132 WTRa
0.002 0.046 0.002 0.217 PRIT-0134 WTRa 0.002 0.059 0.003 0.443
PRIT-0136 WTRa 0.004 0.029 0.014 131.926 PRIT-0127 WTRa 0.014 0.092
0.015 222.339 PRIT-0107 TgRa 1.1 51.0 48.0 >1000 WTRa: Wild Type
Rabbits; TgRa: transgenic rabbits
Example 2: Humanization
Humanization
[0749] Next, the lead candidate PRIT-0128 was subject to
humanization.
[0750] For the identification of a suitable human acceptor
framework during the humanization of the DOTAM binder PRIT-0128, a
combination of two methodologies was used. On the one hand a
classical approach was taken by searching for an acceptor framework
with high sequence homology to the parental antibody and subsequent
grafting of the CDR regions onto this acceptor framework. Each
amino acid difference of the identified frameworks to the parental
antibody was judged for impact on the structural integrity of the
binder and backmutations towards the parental sequence were
introduced whenever appropriate.
[0751] On the other hand, an in house developed in silica tool was
used to predict the orientation of the VH and VL domains of the
humanized versions towards each other (see WO2016/062734). This was
carried out for the virtual grafts of the CDRs on all possible
human germline combinations. The results were compared to the VH-VL
domain orientation of the parental binder to select for framework
combinations which are close in geometry to the starting
antibody.
[0752] In each case the following CDR regions of the parental
antibody were grafted onto the acceptor framework (numbering
according to Kabat):
VH_CDR1: 31-35
VH_CDR2: 50-65
VH_CDR3: 95-102
VL_CDR1: 24-34
VL_CDR2: 50-56
VL_CDR3: 89-97
[0753] The humanization variants were produced in a format
comprising a full length antibody for CEA, with the C-terminus of
one of the heavy chains fused to the N-terminus of the VH domain
the Dotam-binder, and the C-terminus of the other heavy chain fused
to the N-terminus of the VL domain of the DOTAM-binder, forming a
bispecific antibody with two binding sites for CEA and one
functional binding site for DOTAM. Thus, the DOTAM binder was fused
to the C-terminus of the Fc of the tumour targeting IgG as an VH/VL
Fv fusion (without CH1 and Ck respectively). The parental DOTAM
binder PRIT-0128 derived molecule in this bispecific format is
called PRIT-0156.
[0754] Herceptin framework was included as well due to the
suitability in terms of VH/VL prediction and the increased
stability of the framework. For all VH humanized variants, the
human J element hJH2 was used. For all VK humanized variants, the
human J element hJK4 was used.
[0755] The HC4 is a grafting of PRIT-128 on the human germline
IGHV3-30-02 with one backmutation Kabat A49G.
[0756] To get the variable heavy chain HC5, the CDRs were grafted
on human germline hVH_2_26 with A49G as a backmutation and the
deletion of the first amino acid to reflect the original rabbit
N-terminus.
[0757] Grafted on Herceptin V-region (derived from human germline
hVH3_66), the variant HC7 is characterized by a few modifications
in the acceptor framework: deletion of N-terminus E, A49G, A71R,
and S93A.
[0758] For HC10, the CDRs of PRIT-128 were grafted on the human
germline IGHV4_34_01.
[0759] Here, the N-terminus was modified, starting with V2, to
reflect the original rabbit antibody starting with Q2. In addition,
G29F and F31L were considered as backmutation wrt Kabat
nomenclature as well as V71R and F78V in framework 3.
[0760] For the light chain LC1, the CDRs were grafted on the human
germline IGKV1_39_01 without any backmutation. The start was chosen
as 12 to reflect the original rabbit Ab starting with A2.
[0761] The light chain variant LC3 was obtained by grafting of the
CDRs on human germline hVK1_5. D1 was deleted and a I2A
backmutation was considered as a new N-terminus. As additional
backmutations, K42Q and A43P were taken into account.
[0762] Not all possible combinations of the humanization matrix
were produced but a selection of defined combinations was chosen
based on considerations like VH/VL prediction and sequence risks of
the given combination.
Selection of Candidates
[0763] The goal of the humanization was to obtain humanized binders
which do not lose more than a factor of 10 in terms of affinity to
DOTAM. This was achieved with several binders of comparable or even
better affinity to DOTAM.
[0764] As mentioned above, PRIT-0156 is a 2:1 antibody comprising
the rabbit DOTAM binder PRIT-0128 combined with the CEA binder
CH1A1A. PRIT-0178 to PRIT-0204 are humanized variants in the same
format with the same CEA binder. PRIT-0205 up to PRIT-0221
correspond to the PRIT-0178 to PRIT-0204 humanization variants in
the DOTAM binding part, but have the CEA binder changed to
T84.66.
Solution Equilibrium Based kd Determination
[0765] To screen a larger amount of humanization candidates for
their affinity to Pb-DOTAM, solution equilibrium titration (SET)
was used. The table below details the SET based affinity
determination of selected humanized DOTAM binders against Pb-DOTAM.
All antibodies in this table are bispecific antibodies that
comprise bivalent binding to CEA and monovalent binding to Pb-Dotam
(2:1 format):
TABLE-US-00014 Humanized HC/LC Molecule name combination SET
Pb-DOTAM (pM) PRIT-0187-0002 HC10 LC1 0.03 PRIT-0193-0002 HC5 LC3
0.36 PRIT-0195-0004 HC10 LC3 0.40 PRIT-0156-0004 Parental molecule
0.43 PRIT-0182-0002 HC8 LC7 5.54 PRIT-0189-0002 HC7 LC2 5.81
PRIT-0185-0002 HC 2 LC1 5.87 PRIT-0192-0002 HC2 LC3 8.04
PRIT-0183-0004 HC2 LC2 8.11 PRIT-0197-0002 HC7 LC1 8.69
PRIT-0198-0002 HC7 LC3 9.09 PRIT-0182-0004 HC8 LC7 17.14
PRIT-0183-0002 HC2 LC2 22.45 PRIT-0180-0004 HC9 LC6 26.10
PRIT-0190-0002 HC9 LC2 33.83 PRIT-0194-0002 HC8 LC3 34.63
PRIT-0199-0002 HC9 LCI 43.91 PRIT-0180-0002 HC9 LC6 47.70
PRIT-0188-0002 HC4 LC2 54.34 PRIT-0178-0004 HC1 LC6 60.06
PRIT-0179-0004 HC4 LC6 64.92 PRIT-0181-0002 HC4 LC7 65.11
PRIT-0179-0002 HC4 LC6 65.81 PRIT-0178-0002 HC1 LC6 66.68
PRIT-0187-0004 HC10 LC1 78.09 PRIT-0184-0002 HC1 LC1 80.56
PRIT-0200-0002 HC9 LC3 83.24 PRIT-0191-0002 HC1 LC3 111.10
Kinexa Based kd Determination
[0766] For more detailed analysis and an orthogonal method for
affinity determination, Kinexa was used.
Instrumentation and Materials
[0767] A KinExA 3200 instrument from Sapidyne Instruments (Boise,
Id.) with autosampler was used. Polymethylmethacrylate (PMMA) beads
were purchased from Sapidyne, whereas PBS (phosphate buffered
saline), BSA (bovine serum albumin fraction V) and the anti-DOTAM
antibodies were prepared in-house (Roche).
Dylight650.RTM.-conjugated affinity-purified goat anti-human IgG-Fc
Fragment cross-adsorbed antibody was purchased from Bethyl
Laboratories (Montgomery, Tex.). The biotinylated Pb-DOTAM antigens
(Pb-DOTAM-alkyl-biotin isomer A and B,
Pb-DOTAM-Bn-biotin/TCMC-Pb-dPEG3-Biotin, isomer A and B) and the
non-biotinylated Pb-DOTAM were obtained from AREVA Med (Bethesda,
Md.).
Preparation of Antigen Coated Beads
[0768] PMMA beads were coated according to the KinExA Handbook
protocol for biotinylated molecules (Sapidyne). Briefly, first, 10
.mu.g of Biotin-BSA (Thermo Scientific) in 1 ml PBS (pH7.4) was
added per vial (200 mg) of beads for adsorption coating. After
rotating for 2 h at room temperature, the supernatant was removed
and beads were washed 5 times with 1 ml PBS. Second, 1 ml of 100
.mu.g of NeutrAvidin Biotin-Binding Protein (Thermo Scientific) in
PBS containing 10 mg/ml BSA was added to the beads and incubated at
room temperature for additional 2 h to couple NeutrAvidin to the
beads and to provide additional biotin binding sites for subsequent
binding of biotinylated proteins. The NeutrAvidin-coated-beads were
then rinsed 5 times with 1 ml PBS. Finally, the beads were coated
with 200 ng/ml biotinylated Pb-DOTAM-Isomer Mix (50 ng for each
Isomer) in PBS and incubated for further 2 h at room temperature.
Beads were then resuspended in 30 ml PBS and used immediately.
KinExA Equilibrium Assays
[0769] All KinExA experiments were performed at room temperature
(RT) using PBS pH 7.4 as running buffer. Samples were prepared in
running buffer supplemented with 1 mg/ml BSA ("sample buffer"). A
flow rate of 0.25 ml/min was used. A constant amount of anti-DOTAM
antibody with 5 pM binding site concentration was titrated with
Pb-DOTAM antigen by twofold serial dilution starting at 100 pM
(concentration range 0.049 pM-100 pM). One sample of antibody
without antigen served as 100% signal (i.e. without inhibition).
Antigen-antibody complexes were incubated at RT for at least 24 h
to allow equilibrium to be reached. Equilibrated mixtures were then
drawn through a column of Pb-DOTAM-coupled beads in the KinExA
system at a volume of 5 ml permitting unbound antibody to be
captured by the beads without perturbing the equilibrium state of
the solution. Captured antibody was detected using 250 ng/ml
Dylight 6500-conjugated anti-human Fc-fragment specific secondary
antibody in sample buffer. Each sample was measured in duplicates
for all equilibrium experiments.
[0770] The KD was obtained from non-linear regression analysis of
the data using a one-site homogeneous binding model contained
within the KinExA software (Version 4.0.11) using the "standard
analysis" method. The software calculates the KD and determines the
95% confidence interval by fitting the data points to a
theroretical KD curve. The 95% confidence interval (Sapidyne
TechNote TN207R0) is given as KD low and KD high.
Thermostability Measurements for Humanized PRIT Molecules
Method and Data Analysis
[0771] Different variants of the humanized PRIT molecules in the
final format (in 20 mM Histidine, 140 mM NaCl, pH 6.0) were diluted
in the same buffer to 1 mg/ml. 30 .mu.l of each sample was
transferred into a 384-well plate filter device (alongside an
anti-HER3 antibody as reference). After centrifugation at 1,000 g
for 1 min wells were overlaid with 10 .mu.l of paraffin oil. The
plate was centrifuged again (1,000 g for 1 min) and transferred
into the DLS pklate reader (Dyna Pro PlateReader-II, Wyatt).
Starting at 25.degree. C. the temperature was increased at a speed
of 0.05.degree. C./min to 79.9.degree. C. Scattered light was
recorded using the Dynamics Software (V7.0).
[0772] Data was transferred to Excel (Microsoft), sorted by sample
and temperature and a software add-in used to create melting
curves. The temperature, where clear deviation from the baseline
occurred, was defined as "onset of aggregation" and the inflection
point of the melting curve as "melting temperature".
Results
TABLE-US-00015 [0773] Sample onset of aggregation (in .degree. C.)
melting temperature (in .degree. C.) Her 3 63 .+-. 1 67.5 .+-. 1
PRIT-0156 45 .+-. 1 51 .+-. 1 (parental molecule with rabbit DOTAM
binder) PRIT 205 51 .+-. 1 51.5 .+-. 1 PRIT 206 51 .+-. 1 58.1 .+-.
1 PRIT 207 49 .+-. 1 58.1 .+-. 1 PRIT 208 54 .+-. 1 57.7 .+-. 1
PRIT 209 54 .+-. 1 57.7 .+-. 1 PRIT-0213 54 .+-. 1 57.7 .+-. 1
Properties of Candidates
[0774] The tables below summarize the identity of various PRIT
molecules and compare their properties. Preferred compounds were
PRIT-0213 and PRIT-0214.
Summary of Candidates
TABLE-US-00016 [0775] CEA Antibody binder Dotam binder Format PRIT
0218 none WT Rabbit antibody PRIT0156 CHIA1A PRIT-0218 2:1 antibody
with PRIT 0128 dotam binder PRIT 0186 CH1A1A HC5/LC1 (humanized
PRIT- 2:1 format 0218) PRIT-0213 T84.86 HC5/LC1 (humanized PRIT-
2:1 format 0218) PRIT-0187 CH1A1A HC10/LC1 (humanized PRIT- 2:1
format 0218) PRIT-0214 T84.86 HC10/LC1 (humanized PRIT- 2:1 format
0218) PRIT-0206 T84.86 HC5/LC3 (humanized PRIT- 2:1 format 0218)
PRIT-0216 T84.86 HC5/LC3 (humanized PRIT- 2:1 format 0218)
PRIT-0217 T84.86 HC5/LC3 (humanized PRIT- 2:1 format 0218)
PRIT-0208 T84.86 HC7/LC1 (humanized PRIT- 2:1 format 0218)
Comparison of Properties
TABLE-US-00017 [0776] SET Kinexa CEA DOTAM PbDOTAM PbDOTAM Yield T
Agg BiCEAnder Binder [PM] [PM] [mg/l] 266 nm Humanness
Biodistribution PRIT-0156 CH1A1A WT Rabbit 0.01 0.25 25 45 -- -- Ok
PRIT-0186 CH1A1A HC5/LC1 0.4 0.92 38 50 HC LC Ok 58% 57% PRIT-0213
T84.66 HC5/LC1 0.4 0.84 4.0 54 HC LC Ok 58% 57% PRIT-0187 CH1A1A
HC10/ LC1 0.03 0.99 17.8 57 HC LC Ok 40% 57% PRIT-0214 T84.66
HC10/LC1 0.03 0.84 6.0 55 HC LC Ok 40% 57% PRIT-0206 T84.66 HC5/LC3
0.36 1.0 1.32 50 HC LC Ok 58% 55% PRIT-0216 T84.66 HC5/LC3 -- 51
7.0 50 HC LC Ok 58% 55% PRIT-0217 T84.66 HC5/LC3 -- 3 10.0 54 HC LC
Ok 58% 55% PRIT-0208 T84.66 HC7/LC1 8.7 8.3 7.6 59 HC LC Ok 51%
57%
Further data for affinity values as determined by Kinexa are
provided below, for PRIT-0213, (PRIT-0213 is the same molecule as
PRIT-0186, except for another CEA binding VH/VL.)
PRIT-0213
Metal-DOTAM Chelate Affinities of CEA-DOTAM BsAb
TABLE-US-00018 [0777] Antigen KD [pM] 95% CI[pM] Pb-DOTAM 0.84
0.44-1.4 Ca-DOTAM 0.95 0.43-1.7 Bi-DOTAM 5.7 4.6-6.2 Cu-DOTAM
122000 60000-206000
Additional values are as shown below:
TABLE-US-00019 95% confidence Name Antigen KD [pM] internal [pM]
PRIT-0213 Ca-DOTAM 0.95 0.43-1.7* PRIT-0214 Ca-DOTAM 0.52 0.34-0.74
PRIT-0213 Bi-DOTAM 5.7 4.6-6.2* PRIT-0214 Bi-DOTAM 6.0 5.5-6.4
PRIT-0213 Cu-DOTAM 122000 .sub. 60000-206000*.degree. PRIT-0214
CU-DOTAM 38000 .sub. 19000-63000*.degree. *broad confidence
interval, indicating the measured K.sub.D not as precise
.degree.assay not completely optimized for nM - affinity
Sequences
[0778] Sequences for this example are provided below. PRIT-0213 and
-0214 both have a Pb-DOTAM binding site comprising CDRs of SEQ ID
NO 116-121 below. The sequences of the heavy and light chain
variable of the Pb-DOTAM binding site of PRIT-0213 are shown in SEQ
ID NO: 122-123 and those of PRIT-0214 are shown in SEQ ID NO:
124-125.
PRIT-0214 is composed of:
[0779] i) a first heavy chain having the amino acid sequence of SEQ
ID NO: 126;
[0780] ii) a second heavy chain having the amino acid sequence of
SEQ ID NO: 127; and
[0781] iii) two antibody light chains having the amino acid
sequence of SEQ ID NO: 128.
PRIT-0213 is composed of:
[0782] i) a first heavy chain having the amino acid sequence of SEQ
ID NO: 129;
[0783] ii) a second heavy chain having the amino acid sequence of
SEQ ID NO: 130; and
[0784] iii) two antibody light chains having the amino acid
sequence of SEQ ID NO: 128.
TABLE-US-00020 SEQ ID NO Description SEQUENCE 116 heavy chain CDR1
<Pb- gfslstysms Dotam> PRIT-0213 117 heavy chain CDR2 <Pb-
figsrgdtyyaswakg Dotam> PRIT-0213 118 heavy chain CDR3 <Pb-
erdpygggaypphl Dotam> PRIT-0213 119 light chain CDR1 <Pb-
qsshsvysdndla Dotam> PRIT-0213 120 light chain CDR2 <Pb-
qasklas Dotam> PRIT-0213 121 light chain CDR3 <Pb-
lggyddesdtyg Dotam> PRIT-0213 122 heavy chain variable
vtlkesgpvl vkptetltlt ctvsgfslst domain 1 of <Pb-Dotam>
ysmswirqpp gkalewlgfi gsrgdtyyas wakgrltisk PRIT-0213 dtsksqvvlt
mtnmdpvdta tyycarerdp ygggaypphl wgrgtlvtvs s 123 light chain
variable iqmtqspssl sasvgdrvti tcqsshsvys domain <Pb-Dotam>
dndlawyqqk pgkapklliy qasklasgvp srfsgsgsgt dftltissiq PRIT-0213
pedfatyycl ggyddesdty gfgggtkvei k 124 heavy chain variable
vqlqqwgagl lkpsetlslt cavygfslst domain <Pb-Dotam> ysmswirqpp
gkglewigfi gsrgdtyyas wakgrvtisr dtsknqvslk PRIT-0214 lssvtaadta
vyycarerdp ygggaypphl wgrgtlvtvs s 125 light chain variable
iqmtqspssl sasvgdrvti tcqsshsvys domain <Pb-Dotam> dndlawyqqk
pgkapklliy qasklasgvp srfsgsgsgt dftltisslq PRIT-0214 pedfatyycl
ggyddesdty gfgggtkvei k 126 heavy chain 1 of 1 qvqlvqsgae
vkkpgssvkv sckasgfnik dtymhwvrqa pgqglewmgr bispecific, trivalent
51 idpangnsky vpkfqgrvti tadtststay melsslrsed tavyycapfg
<CEA/Pb-Dotam>PRIT- 101 yyvsdyamay wgqgtlvtvs sastkgpsvf
plapssksts ggtaalgclv 0214 VH_84.66 151 kdyfpepvtv swnsgaltsg
vhtfpavlqs sglyslssvv tvpssslgtq 201 tyicnvnhkp sntkvdkkve
pkscdkthtc ppcpapeaag gpsvflfppk 251 pkdtlmisrt pevtcvvvdv
shedpevkfn wyvdgvevhn aktkpreeqy 301 nstyrvvsvl tvlhqdwlng
keykckvsnk algapiekti skakgqprep 351 qvytlppcrd eltknqvslw
clvkgfypsd iavewesngq pennykttpp 401 vldsdgsffl yskltvdksr
wqqgnvfscs vmhealhnhy tqkslslspg 451 ggggsggggs ggggsggggs
vqlqqwgagl lkpsetlslt cavygfslst 501 ysmswirqpp gkglewigfi
gsrgdtyyas wakgrvtisr dtsknqvslk 551 lssvtaadta vyycarerdp
ygggaypphl wgrgtlvtvs s 127 heavy chain 2 of 1 qvqlvqsgae
vkkpgssvkv sckasgfnik dtymhwvrqa pgqglewmgr bispecific, 51
idpangnsky vpkfqgrvti tadtststay melsslrsed tavyycapfg trivalent
<CEA/Pb-Dotam> 101 yyvsdyamay wgqgtlvtvs sastkgpsvf
plapssksts ggtaalgclv PRIT-0214 VL_84.66 151 kdyfpepvtv swnsgaltsg
vhtfpavlqs sglyslssvv tvpssslgtq 201 tyicnvnhkp sntkvdkkve
pkscdkthtc ppcpapeaag gpsvflfppk 251 pkdtlmisrt pevtcyvvdv
shedpevkfn wyydgvevhn aktkpreeqy 301 nstyrvvsyl tvlhqdwlng
keykckvsnk algapiekti skakgqprep 351 qvctlppsrd eltknqvsls
cavkgfypsd iavewesngq pennykttpp 401 vldsdgsffl vskltvdksr
wqqgnvfscs vmhealhnhy tqkslslspg 451 ggggsggggs ggggsggggs
iqmtqspssl sasvgdrvti tcqsshsvys 501 dndlawyqqk pgkapklliy
qasklasgvp srfsgsgsgt dftltisslq 551 pedfatyycl ggyddesdty
gfgggtkvei k 128 light chain <CEA> 1 eivltqspat lslspgerat
lscragesvd ifgvgflhwy qqkpgqaprl 84.66 51 liyrasnrat giparfsgsg
sgtdftltis slepedfavy ycqqtnedpy 101 tfgqgtklei krtvaapsvf
ifppsdeqlk sgtasvvcll nnfypreakv 151 qwkvdnalqs gnsqesvteq
dskdstysls stltlskady ekhkvyacev 201 thqglsspvt ksfnrgec 129 heavy
chain 1 of 1 qvqlvqsgae ykkpgssvkv sckasgfnik dtymhwvrqa pgqglewmgr
bispecific, trivalent 51 idpangnsky vpkfqgrvti tadtststay
melsslrsed tavyycapfg <CEA/Pb-Dotam> PRIT- 101 yyvsdyamay
wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv 0213 VH_84.66 .fwdarw.
knob 151 kdyfpepvtv swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq 201
tyicnvnhkp sntkvdkkve pkscdkthtc ppcpapeaag gpsvflfppk 251
pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy 301
nstyrvvsvl tvlhqdwlng keykckvsnk algapiekti skakgqprep 351
qvytlppcrd eltknqvslw clvkgfypsd iavewesngq pennykttpp 401
vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg 451
ggggsggggs ggggsggggs vtlkesgpvl vkptetltlt ctvsgfslst 501
ysmswirqpp gkalewlgfi gsrgdtyyas wakgrltisk dtsksqvvlt 551
mtnmdpvdta tyycarerdp ygggaypphl wgrgtlvtvs s 130 heavy chain 2 of
1 qvqlvqsgae vkkpgssvkv sckasgfnik dtymhwvrqa pgqglewmgr
bispecific, trivalent 51 idpangnsky vpkfqgrvti tadtststay
melsslrsed tavyycapfg <CEA/Pb-Dotam> PRIT- 101 yyvsdyamay
wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv 0213 VL_84.66 .fwdarw.
hole 151 kdyfpepvtv swnsgaltsg vhtfpavlqs sglyslssvv tvpssslgtq 201
tyicnvnhkp sntkvdkkve pkscdkthtc ppcpapeaag gpsvflfppk 251
pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy 301
nstyrvvsvl tvlhqdwlng keykckvsnk algapiekti skakgqprep 351
qvctlppsrd eltknqvsls cavkgfypsd iavewesngq pennykttpp 401
vldsdgsffl vskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg 451
ggggsggggs ggggsggggs iqmtqspssl sasvgdrvti tcqsshsvys 501
dndlawyqqk pgkapklliy qasklasgvp srfsgsgsgt dftltisslq 551
pedfatyycl ggyddesdty gfgggtkvei k 131 heavy chain 1 of 1
qvqlvqsgae vkkpgssvkv sckasgfnik dtymhwvrqa pgqglewmgr bispecific,
<CEA/Pb- 51 idpangnsky vpkfqgrvti tadtststay melsslrsed
tavyycapfg Dotam> Rabbit Dotam_ 101 yyvsdyamay wgqgtlvtvs
sastkgpsvf plapssksts ggtaalgclv 84.66 151 kdyfpepvtv swnsgaltsg
vhtfpavlqs sglyslssvv tvpssslgtq 201 tyicnvnhkp sntkvdkkve
pkscdkthtc ppcpapeaag gpsvflfppk 251 pkdtlmisrt pevtcvvvdv
shedpevkfn wyvdgvevhn aktkpreeqy 301 nstyrvvsvl tvlhqdwlng
keykckvsnk algapiekti skakgqprep 351 qvctlppsrd eltknqvsls
cavkgfypsd iavewesngq pennykttpp 401 vldsdgsffl vskltvdksr
wqqgnvfscs vmhealhnhy tqkslslspg 451 ggggsggggs ggggsggggs
avltqtpspv spavggtvti scqsshsvys 501 dndlawyqqk lgqppklliy
qasklasgvs srfsgsgsgt qftltisgvq 551 sddaatyycl ggyddesdty
gfgggtevvv k 132 HC5 VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWI
RQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKD
TSKSQVVLTMTNMDPVDTATYYCARERDPYGGG AYPPHLWGRGTLVTVSS 133 LC1
SIQMTQSPSSLSASVGDRVTITCQSSHSVYSDNDLA
WYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGT
DFTLTISSLQPEDFATYYCLGGYDDESDTYGFGGGT KVEIK 134 LC3 aqmtqspstl
sasvgdrvti tcqsshsvys dndlawyqqk pgqppklliy qasklasgvp srfsgsgsgt
eftltisslq pddfatyycl ggyddesdty gfgggtkvei k 135 HC7 vqlvesgggl
vqpggslrls caasgfslst ysmswvrqap gkglewvgfi gsrgdtyyas wakgrftisr
dtskntaylq mnslraedta vyycarerdp ygggaypphl wgrgtlvtvs s 136 HC10
vqlqqwgagl lkpsetlslt cavygfslst ysmswirqpp gkglewigfi gsrgdtyyas
wakgrvtisr dtsknqvslk lssvtaadta vyycarerdp ygggaypphl wgrgtlvtvs s
137 T84.66 VH 1 qvqlvqsgae vkkpgssvkv sckasgfnik dtymhwvrqa
pgqglewmgr idpangnsky vpkfqgrvti tadtststay melsslrsed tavyycapfg
yyvsdyamay wgqgtlvtvs s 138 T84.66 VL
EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFL
HWYQQKPGQAPRLLIYRASNRATGIPARFSGSGSG
TDFTLTISSLEPEDFAVYYCQQTNEDPYTFGQGTKL EIK 139 CH1A1A VH qvqlvqsgae
vkkpgasvkv sckasgytft efgmnwvrqa pgqglewmgw intktgeaty veefkgrvtf
ttdtststay melrslrsdd tavyycarwd fayyveamdy wgqgttvtvs s 140 CH1A1A
VL diqmtqspss lsasvgdrvt itckasaavg tyvawyqqkp gkapklliys
asyrkrgvps rfsgsgsgtd ftltisslqp edfatyychq yytyplftfg qgtkleik
Example 3: Crystallization, Data Collection and Structure
Determination of the Fab P1AA1227 Pb-DOTAM Complex
[0785] For complex formation the Fab derived from the humanized
VH/VL in PRIT-0213, called P1AA1227, at 26 mg/ml was mixed with
Pb-DOTAM powder in a molar ratio of 1:4.2. After 2 hour incubation
at 4.degree. C. initial crystallization trials were performed in
sitting drop vapour diffusion setups at 21.degree. C. using the
JCSG+screen (Qiagen, Hilden). Crystals appeared within 5 days out
of 0.2 M (NH4)2SO4, 0.1 M BIS-TRIS pH 5.5, 25% w/v PEG3350.
Crystals were harvested directly from the screening plate without
any further optimization step.
[0786] Data collection and structure determination. For data
collection crystals were flash frozen at 100K in precipitant
solution containing 10% ethylenglycol. Diffraction data were
collected at a wavelength of 1.0000 .ANG. using a PILATUS 6M
detector at the beamline X10SA of the Swiss Light Source (Villigen,
Switzerland). Data have been processed with XDS (Kabsch, W. Acta
Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). The
crystals of the complex belong to space group C2 with cell axes of
a=135.63 .ANG., b=56.42 .ANG., c=64.52 .ANG. and
.beta.=108.36.degree. and diffract to a resolution of 1.40 .ANG..
The structure was determined by molecular replacement with PHASER
(McCoy, A. J, Grosse-Kunstleve, R. W., Adams, P. D., Storoni, L.
C., and Read, R. J. J. Appl. Cryst. 40, 658-674 (2007)) using the
coordinates of an in house Fab structure as search model.
Difference electron density was used to place the Pb-DOTAM and to
change amino acids according to the sequence differences by real
space refinement. Structures were refined with programs from the
CCP4 suite (Collaborative Computational Project, Number 4 Acta
Cryst. D50, 760-763 (1994).) and BUSTER (Bricogne, G., Blanc, E.,
Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P.,
Sharff, A., Smart, O. S., Vonrhein, C., Womack, T. O. (2011).
Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing
Ltd.). Manual rebuilding was done with COOT (Emsley, P., Lohkamp,
B., Scott, W. G. and Cowtan, K. Acta Cryst D66, 486-501
(2010)).
[0787] Data collection and refinement statistics are summarized
below.
All graphical presentations were prepared with PYMOL (The Pymol
Molecular Graphics System, Version 1.7.4. Schrodinger, LLC.).
Data Collection and Refinement Statistics for Fab P1AA1227-Pb-DOTAM
Complex
TABLE-US-00021 [0788] P1AA1227-Pb-DOTAM Data collection Space group
C2 Cell dimensions a, b, c (.ANG.) 135.63, 56.42, 64.52 .alpha.,
.beta., .gamma.(.degree.) 90, 108.36, 90 Resolution (.ANG.) 1.40
R.sub.sym or R.sub.merge 0.041 I/.delta.I 10.3 (0.94) Completeness
(%) 94.4 (86.1) Redundancy 3.37 (3.33) Refinement Resolution
(.ANG.) 48.9-1.40 No. reflections 81631 R.sub.work/R.sub.free
19.21/22.38 No. atoms Protein 3342 Water 523 Pb-DOTAM 29 B-factors
Protein 14.81 Water 37.46 Pb-DOTAM 21.09 R.m.s. deviations Bond
lengths (.ANG.) 0.011 Bond angles ( .degree.) 1.57 *Values in
parentheses are for highest-resolution shell.
Structure of Fab P1AA1227 in Complex with Pb-DOTAM
[0789] In order to characterize the interaction details of the
Pb-DOTAM with Fab P1AA1227 we determined the crystal structure of
the complex at a resolution of 1.40 .ANG.. The structure reveals
Fab P1AA1227 to bind to Pb-DOTAM by main contributions of the CDR1
and CDR3 of the light chain and by CDR2 and CDR3 of the heavy
chain.
[0790] Analysis of the binding interface with the program PISA
reveals an interaction pattern of Fab P1AA1227 with the Pb-DOTAM
via 3 hydrogen bonds, polar interactions and van der Waals
contacts. The Pb-DOTAM is bound in a pocket formed by heavy and
light chain. This pocket has the shape of a box which is open on
one side. Side walls and bottom of the pocket contribute apolar
interactions whereas at the rim of the walls polar interactions
dominate. Side chain hydrogen bonds are formed between CDR3
residues of heavy chain Glu95 and Asp97 with DOTAM carbamoyl
nitrogen atoms N7 and N8. An additional hydrogen bond is
established via the main chain carbonyl atom of Arg96 with atom N7
of DOTAM. The complex is further stabilized through apolar
interactions of heavy chain CDR2 Phe50 and Tyr58 side chains which
are oriented edge to face to the azacyclododecane ring. The light
chain contributes mostly the "bottom" of the pocket with CDR3
residues Gly91-Tyr96 providing apolar contacts to the
tetracyclododecane ring. Asp32 entertains a hydrogen bond to the
carbamoyl nitrogen atom N6 of DOTAM. (Numbering according to
Kabat).
[0791] The table below shows the heavy chain paratope residues,
based on analysis with the program PISA.
TABLE-US-00022 Heavy Chain Residues (Kabat number) Type of
interaction Pb-DOTAM Phe50 apolar Edge to face to azacyclodo-
decane ring Asp56 polar N8 Tyr58 apolar Edge to face to azacyclodo-
decane ring G1u95 H-bond N7 Arg96 H-bond (mc carbonyl) N7 Asp97
H-bond N8 Pro98 Edge-face Above Pb-Dotam, 4.ANG. distance Tyr99
polar (but distance 6A) N5 Ala100C apolar C12 Tyr 100D polar (mc
atoms) N7
The Table below shows the light chain paratope residues, based on
analysis with the program PISA.
TABLE-US-00023 Light chain residues (Kabat number) Type of
interaction Pb-DOTAM Tyr28 apolar Edge to face to azacyclododecane
ring Asp32 polar, H-bond N6 G1y91 apolar below azacyclododecane
ring Tyr92 apolar below azacyclododecane ring Asp93 apolar below
azacyclododecane ring Thr95C apolar below azacyclododecane ring
Tyr96 apolar Edge to face to azacyclododecane ring
Paratope residues are also underlined in the sequences below:
TABLE-US-00024 >P1AA1227_HC (SEQ ID NO.: 145)
VTLKESGPVLVKPTETLTLTCTVSGFSLSTYSMSWIRQPPGKALEWLGF
IGSRGDTYYASWAKGRLTISTSKSQVVLTMTNMDPVDTATYYCARERDP
YGGGAYPPHLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHICPSNTKVDKKVEPKSC >P1AA1227_LC (SEQ ID NO.: 146)
SIQMTQSPSSLSASVGDRVTITCQSSHSVYSDNDLAWYQQKPGKAPKLL
IYQASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGGYDDES
DTYGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSISTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
Example 4: Generation of CEA-Split-DOTAM VD/VL Antibodies
[0792] Methods of PRIT (Pretargeted radioimmunotherapy) using
bispecific antibodies having a binding site for the target antigen
and a binding site for the radiolabelled compound commonly use a
clearing agent (CA) between the administrations of antibody and
radioligand, to ensure effective targeting and high
tumour-to-normal tissue absorbed dose ratios (see FIG. 3). In an
example of one such method, injected BsAb is allowed sufficient
time for penetrating into the tumours, generally 4-10 days, after
which circulating BsAb is neutralized using a Pb-DOTAM-dextran-500
CA. The CA blocks .sup.212Pb-DOTAM binding to nontargeted BsAb
without penetrating into the tumour, which would block the
pretargeted sites. This pretargeting regimen allows efficient
tumour accumulation of the subsequently administered radiolabelled
chelate, .sup.212Pb-DOTAM.
[0793] However, in methods involving a clearing agent, the use of a
CA introduces a further step to the method which is inefficient.
Moreover, it can be important to choose the timing and dosing of
the CA administration with care, which is a complicating
factor.
[0794] To address the problems associated with use of a clearing
agent, the present inventors have proposed a strategy of splitting
the DOTAM VL and VH domains, such that they are found on separate
antibodies.
[0795] The generation of exemplary split DOTAM VH/VL antibodies is
discussed further below
Generation of Plasmids for the Recombinant Expression of Antibody
Heavy or Light Chains
[0796] Desired proteins were expressed by transient transfection of
human embryonic kidney cells (HEK 293). For the expression of a
desired gene/protein (e.g. full length antibody heavy chain, full
length antibody light chain, or a full length antibody heavy chain
containing an additional domain (e.g. an immunoglobulin heavy or
light chain variable domain at its C-terminus) a transcription unit
comprising the following functional elements was used: [0797] the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0798] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0799] a
murine immunoglobulin heavy chain signal sequence (SS), [0800] a
gene/protein to be expressed, and [0801] the bovine growth hormone
polyadenylation sequence (BGH pA).
[0802] In addition to the expression unit/cassette including the
desired gene to be expressed the basic/standard mammalian
expression plasmid contained [0803] an origin of replication from
the vector pUC18 which allows replication of this plasmid in E.
coli, and [0804] a beta-lactamase gene which confers ampicillin
resistance in E. coli.
a) Expression Plasmid for Antibody Heavy Chains
[0805] Antibody heavy chain encoding genes including C-terminal
fusion genes comprising a complete and functional antibody heavy
chain, followed by an additional antibody V-heavy or V-light domain
was assembled by fusing a DNA fragment coding for the respective
sequence elements (V-heavy or V-light) separated each by a G4Sx4
linker to the C-terminus of the CH3 domain of a human IgG molecule
(VH-CH1-hinge-CH2-CH3-linker-VH or VH-CH1-hinge-CH2-CH3-linker-VL).
Recombinant antibody molecules bearing one VH and one VL domain at
the C-termini of the two CH3 domains, respectively, were expressed
using the knob-into-hole technology.
[0806] The expression plasmids for the transient expression of an
antibody heavy chain with a C-terminal VH or VL domain in HEK293
cells comprised besides the antibody heavy chain fragment with
C-terminal VH or VL domain expression cassette, an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli. The transcription unit of the
antibody heavy chain fragment with C-terminal VH or VL domain
fusion gene comprises the following functional elements: [0807] the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0808] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0809] a
murine immunoglobulin heavy chain signal sequence, [0810] an
antibody heavy chain (VH-CH1-hinge-CH2-CH3-linker-VH or
VH-CH1-hinge-CH2-CH3-linker-VL) encoding nucleic acid, and [0811]
the bovine growth hormone polyadenylation sequence (BGH pA).
[0812] The amino acid sequence of the mature antibody heavy chain
fragment with C-terminal VH or VL domain fusion protein is shown
below:
TABLE-US-00025 PRIT Split Antibody with DOTAM-VH-P1AD8749
>D1AC4022 (SEQ ID NO.: 28) (SEQ ID NO.: 99) (SEQ ID NO.: 100)
QVQLVQSGAEVICKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGW
INTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWD
FAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK
PICDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >D1AA4507
(SEQ ID NO.: 32) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGW
INTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWD
FAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
GGGGSGGGGSGGGGSGGGGSVTLKESGPVLVKPTETLTLTCTVSGFSLST
YSMSWIRQPPGKALEWLGFIGSRGDTYYASWAKGRLTISKDTSKSQVVLT
MTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTLVTVSS PRIT split antibody with
DOTAM-VL-P1AD8592 >:D1AA4506 (SEQ ID NO.: 33)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGW
INTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWD
FAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
GGGGSGGGGSGGGGSGGGGSIQMTQSPSSLSASVGDRVTITCQSSHSVYS
DNDLAWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCLGGYDDESDTYGFGGGTKVEIK >:D1AC4023 (SEQ ID NO.: 30, 97)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGW
INTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWD
FAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
b) Expression Plasmid for Antibody Light Chains
[0813] Antibody light chain encoding genes comprising a complete
and functional antibody light chain was assembled by fusing a DNA
fragment coding for the respective sequence elements.
[0814] The expression plasmid for the transient expression of an
antibody light chain comprised besides the antibody light chain
fragment an origin of replication from the vector pUC18, which
allows replication of this plasmid in E. coli, and a beta-lactamase
gene which confers ampicillin resistance in E. coli. The
transcription unit of the antibody light chain fragment comprises
the following functional elements: [0815] the immediate early
enhancer and promoter from the human cytomegalovirus (P-CMV)
including intron A, [0816] a human heavy chain immunoglobulin
5'-untranslated region (5'UTR), [0817] a murine immunoglobulin
heavy chain signal sequence, [0818] an antibody light chain (VL-CL)
encoding nucleic acid, and [0819] the bovine growth hormone
polyadenylation sequence (BGH pA).
[0820] The amino acid sequence of the mature antibody light chain
fragment is the same for both P1AD8592 and P1AD8749.
TABLE-US-00026 >D1AA3384 (SEQ ID NO: 34) (SEQ ID NO.: 103) (SEQ
ID NO.: 115) DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKWYS
ASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFG
QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC,,
Transient Expression of the Antibody Molecules
[0821] The antibody molecules were generated in transiently
transfected HEK293 cells (human embryonic kidney cell line
293-derived) cultivated in F17 Medium (Invitrogen Corp.). For
transfection "293-Free" Transfection Reagent (Novagen) was used.
The respective antibody heavy- and light chain molecules as
described above were expressed from individual expression plasmids.
Transfections were performed as specified in the manufacturer's
instructions. Immunoglobulin-containing cell culture supernatants
were harvested three to seven (3-7) days after transfection.
Supernatants were stored at reduced temperature (e.g. -80.degree.
C.) until purification.
[0822] General information regarding the recombinant expression of
human immunoglobulins in e.g. HEK293 cells is given in: Meissner,
P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
[0823] The PRIT Hemibodies (split antibodies) were purified by a
MabSelect Sure (Affinity Chromatography) and followed by Superdex
200 (Size Exclusion Chromatography). For the PRIT Split Antibody
with DOTAM-VL-P1AD8592 5 mg with a concentration of 1.372 mg/mL and
a purity >96% based on analytical SEC and CE-SDS were produced.
For the PRIT Split Antibody with DOTAM-VH-P1AD8749 14 mg with a
concentration of 2.03 mg/mL and a purity >91% based on
analytical SEC and CE-SDS were produced.
[0824] Antibodies P1AE4956 and P1AE4957 were also generated and
their sequences are provided herein. (P1AE4956 has heavy chains of
SEQ ID NO: 51 and 52 and the light chain of SEQ ID NO: 54; P1AE4957
has heavy chains of SEQ ID NO 55 and 56 and the light chain of SEQ
ID NO: 58). For the PRIT Split Antibody with DOTAM-VL-P1AE4957 19
mg with a concentration of 2.6 mg/mL and a purity >81.6% based
on analytical SEC and CE-SDS were produced. For the PRIT Split
Antibody with DOTAM-VH-P1AE4956 6.9 mg with a concentration of 1.5
mg/mL and a purity >90% based on analytical SEC and CE-SDS were
produced. ESI-MS was used too confirm the identity of the PRIT
hemibodies.
Example 5: FACS Analysis of Split Antibody Functionality
[0825] To assess the functionality of the split antibodies or
hemibodies, MKN-45 cells were detached from the culture vessel
using accutase at 37.degree. C. for 10 minutes. Subsequently, the
cells were washed twice in PBS, and seeded into 96 well v-bottom
plates to a final density of 4.times.10.sup.6 cells/well.
[0826] The hemibodies P1AD8749 and P1AD8592 and a human ISO control
were mixed 1:1 added to the cells in concentrations as indicated in
FIG. 5. Subsequently, the cells were incubated for 1 h on ice and
washed twice in PBS. The cell pellet was resuspended and 40
.mu.l/well of detection reagent was added, either <human
IgG(H+L)>FITC, (10 .mu.g/ml) or Pb_Dotam_FITC 1:100=>(10
.mu.g/ml) in PBS/5% FCS. After 60 min incubation on ice, the cells
were washed twice in PBS and resuspended in 200 .mu.l PBS/5% FCS
for measurement of FITC fluorescence using a FACS canto.
[0827] To assess the binding capability of the hemibodies to CEA on
MKN-45 cells, they were detected using of antibodies using human
IgG specific secondary antibodies (FIG. 5). As expected, no
significant binding of the human ISO control is observed on these
cells. When adjusted to the same IgG concentration, both hemibodies
as well as the combination of both shows a dose dependent binding
to MKN-45 cells, with a pronounced hook effect at very high
concentrations as expected. This experiment demonstrates that the
CEA binding is functional in the hemibodies.
[0828] To assess the binding capability of the hemibodies to DOTAM,
they were bound to the cells either in the presence of a human ISO
control or their respective split antibody partner in a 1:1 ratio.
After their binding to MKN-45 cells, the cells were washed to
remove unbound antibody. Subsequently, Pb-DOTAM-FITC (fluorescently
labelled Pb-DOTAM) was added to detect DOTAM binding competent cell
bound antibodies (FIG. 6). As expected, no significant FITC is
observed on these cells when one of the split antibody partners is
combined with the of the human ISO control. Only a combination of
both hemibodies in a 1:1 ratio shows a dose dependent FITC signal.
This experiment shows that the DOTAM binding site becomes
functional when both hemibodies come together on one cell.
Example 6: In Vivo Studies
Example 6a: Materials and Methods--General
[0829] All experimental protocols were reviewed and approved by the
local authorities (Comite Regional d'Ethique de l'Experimentation
Animale du Limousin [CREEAL], Laboratoire Departemental d'Analyses
et de Recherches de la Haute-Vienne). Female severe combined
immunodeficiency (SCID) mice (Charles River) were maintained under
specific and opportunistic pathogen free (SOPF) conditions with
daily cycles of light and darkness (12 h/12 h), in line with
ethical guidelines. No manipulations were performed during the
first 5 days after arrival, to allow the animals to acclimatize to
the new environment. Animals were controlled daily for clinical
symptoms and detection of adverse events.
[0830] Solid xenografts were established by subcutaneous (SC)
injection of CEA-expressing tumor cells in cell culture media mixed
1:1 with Corning.RTM. Matrigel.RTM. basement membrane matrix
(growth factor reduced; cat No. 354230). Tumor volumes were
estimated through manual calipering 3 times per week, calculated
according to the formula:
volume=0.5.times.length.times.width.sup.2. Additional tumor
measurements were made as needed depending on the tumor growth
rate.
[0831] Mice were euthanized before the scheduled endpoint if they
showed signs of unamenable distress or pain due to tumor burden,
side effects of the injections, or other causes. Indications of
pain, distress, or discomfort include, but are not limited to,
acute body weight (BW) loss, scruffy fur, diarrhea, hunched
posture, and lethargy. The BW of treated animals was measured 3
times per week, with additional measurements as needed depending on
the health status. Wet food was provided to all mice starting the
day after the radioactive injection, for 7 days or until all
individuals had recovered sufficiently from any acute BW loss. Mice
whose BW loss exceeded 20% of their initial BW or whose tumor
volume reached 3000 mm.sup.3 were euthanized immediately. Other
factors taken into account for euthanasia for ethical reasons were
tumor status (e.g. necrotic areas, blood/liquid leaking out, signs
of automutilation) and general appearance of the animal (e.g. fur,
posture, movement).
[0832] To minimize re-ingestion of radioactive urine/feces, all
efficacy study mice were placed in cages with grilled floors for 4
hours after .sup.212Pb-DOTAM administration, before being
transferred to new cages with standard bedding. All cages were then
changed at 24 hours post injection (p.i.). This procedure was not
performed for mice sacrificed for biodistribution purposes within
24 hours after the radioactive injection.
[0833] Blood was collected at the time of euthanasia from the
venous sinus using retro-orbital bleeding on anesthetized mice,
before termination through cervical dislocation followed by
additional tissue harvest for radioactive measurements and/or
histological analysis, as mandated by the protocols. Unexpected or
abnormal conditions were documented. Tissues collected for formalin
fixation were immediately put in 10% neutral buffered formalin
(4.degree. C.) and then transferred to phosphate-buffered saline
(PBS; 4.degree. C.) after 5 days. Organs and tissues collected for
biodistribution purposes were weighed and measured for
radioactivity using a 2470 WIZARD.sup.2 automatic gamma counter
(PerkinElmer), and the percent injected dose per gram of tissue (%
ID/g) subsequently calculated, including corrections for decay and
background.
[0834] Statistical analysis was performed using GraphPad Prism 7
(GraphPad Software, Inc.) and JMP 12 (SAS Institute Inc.). Curve
analysis of tumor growth inhibition (TGI) was performed based on
mean tumor volumes using the formula:
T .times. G .times. I = 100 - v treatment , d - v treatmet , 0 _ v
ref , d - v ref , 0 _ .times. 100 ##EQU00001##
where d indicates study day and 0 the baseline value. Vehicle was
selected as the reference group. Tumor regression (TR) was
calculated according to:
T .times. R = v treatment , 0 - v treatment , d _ v treatment , 0
##EQU00002##
where positive values indicated tumor regression, and values below
-1 growth beyond the double baseline value.
Test Compounds
[0835] The compounds utilized in the described studies are
presented in the tables below, respectively for bispecific
antibodies, clearing agents, and radiolabeled chelates.
[0836] CEA-DOTAM (R07198427, PRIT-0213) is a fully humanized BsAb
targeting the T84.66 epitope of CEA, whereas DIG-DOTAM (R07204012)
is a non-CEA-binding BsAb used as a negative control. P1 AD8749, P1
AD8592, P1AE4956, and P1AE4957 are CEA-split-DOTAM-VH/VL antibodies
targeting the CH1A1A or A5B7 epitopes of CEA. All antibody
constructs were stored at -80.degree. C. until the day of injection
when they were thawed and diluted in standard vehicle buffer (20 mM
Histidine, 140 mM NaCl; pH 6.0) or 0.9% NaCl to their final
respective concentrations for intravenous (IV) or intraperitoneal
(IP) administration.
[0837] The Pb-DOTAM-dextran-500 CA (R07201869) was stored at
-20.degree. C. until the day of injection when it was thawed and
diluted in PBS for IV or IP administration.
[0838] The DOTAM chelate for radiolabeling was provided by
Macrocyclics and maintained at -20.degree. C. before radiolabeling,
performed by Orano Med (Razes, France). .sup.212Pb-DOTAM
(R07205834) was generated by elution with DOTAM from a thorium
generator, and subsequently quenched with Ca after labeling. The
.sup.212Pb-DOTAM solution was diluted with 0.9% NaCl to obtain the
desired .sup.212Pb activity concentration for IV injection. Mice in
vehicle control groups received multiple injections of vehicle
buffer instead of BsAb, CA, and .sup.212Pb-DOTAM.
Bispecific Antibodies
TABLE-US-00027 [0839] Compound Target Protocols CEA-DOTAM T84.66
144, 158, 160 (RO7198427, PRIT- 0213 ) DIG-DOTAM Digoxigenin 160
(RO7204012) CEA-split-DOTAM-VH CH1A1A 144, 158 P1AD8749
CEA-split-DOTAM- CH1A1A 175, 185, 189 VH-AST PlAF0171
CEA-split-D-OTAM-VL CH1A1A 144, 158 P1AD8592 175, 185, 189
CEA-split-DOTAM-VH A5B7 158 PlAE4956 CEA-split-DOTAM-VL A5B7 158
PlAE4957 CEA-split-DOTAM- T84.66 185, 189 VH-AST PlAF0298
CEA-split-DOTAM-VL T84.66 185, 189 PlAF0709
Clearing Agents
TABLE-US-00028 [0840] Compound Protocols Ca-DOTAM-dextran-500 144,
158, 160 (RO7201869)
Radiolabeled Chelates
TABLE-US-00029 [0841] Compound Quenching Protocols .sup.212Pb-DOTAM
Ca 144, 158, (RO7205834) 160, 175, 185, 189 .sup.212Pb-DOTAM-CEA-
Ca 160 DOTAM
Tumor Models
[0842] The tumor cell line used and the injected amount for
inoculation in mice is described in the table below. BxPC3 is a
human primary pancreatic adenocarcinoma cell line, naturally
expressing CEA. Cells were cultured in RPMI 1640 Medium,
GlutaMAX.TM. Supplement, HEPES (Gibco, ref. No. 72400-021) enriched
with 10% fetal bovine serum (GE Healthcare Hyclone SH30088.03).
Solid xenografts were established in each SCID mouse on study day 0
by subcutaneous injection of cells in RPMI media mixed 1:1 with
Corning.RTM. Matrigel.RTM. basement membrane matrix (growth factor
reduced; cat No. 354230), into the right flank.
Tumor Cell Lines
TABLE-US-00030 [0843] Cell line Cells per mouse Injected volume
Protocols Supplier BxPC3 5.times.10.sup.6 100 .mu.L 144, 158, 160,
ECACC* 175, 185, 189 *European Collection of Authenticated Cell
Cultures (Salisbury, UK)
Example 6b: Protocol 144
[0844] The aim of protocol 144 was to provide PK and in vivo
distribution data of pretargeted .sup.212Pb-DOTAM in SCID mice
carrying SC BxPC3 tumors after 2-step PRIT using
CEA-split-DOTAM-VH/VL BsAbs.
[0845] Two-step PRIT was performed by injection of the
CEA-split-DOTAM-VH and CEA-split-DOTAM-VL (P1AD8749 and P1AD8592),
separately or together, followed 7 days later by .sup.212Pb-DOTAM.
Mice were sacrificed 6 hours after the radioactive injection, and
blood and organs harvested for radioactive measurement. The 2-step
scheme was compared with 3-step PRIT using the standard CEA-DOTAM
bispecific antibody, followed 7 days later by Ca-DOTAM-dextran-500
CA, and .sup.212Pb-DOTAM 24 hours after the CA.
[0846] PK data of CEA-split-DOTAM-VH/VL clearance was collected by
repeated blood sampling from 1 hour to 7 days after the antibody
injection, and subsequently analyzed by an ELISA.
[0847] The study outline is shown in FIG. 7. FIG. 7a shows the
outline of the 2-step PRIT regimen, including blood sampling for
CEA-split-DOTAM-VH/VL PK, in SCID mice carrying SC BxPC3 tumors.
FIG. 7b shows the outline of the 3-step PRIT regimen, performed in
SCID mice carrying SC BxPC3 tumors (h=hours, d=days).
Study Design
[0848] The time course and design of protocol 144 is shown in the
tables below.
Time Course of Protocol 144
TABLE-US-00031 [0849] Study day Date Experimental procedure 0 2018
Apr. 2 Preparation of BxPC3 cells and filling of syringes 0 2018
Apr. 2 SC injection of BxPC3 cells 14 2018 Apr. 16 IV injection of
CEA-DOTAM BsAb (group D) 15 2018 Apr. 17 IV injection of
CEA-split-DOTAM-VH/VL BsAbs (groups Aa, Ab, Ba, Bb, Ca, Cb) 15 2018
Apr. 17 Retro-orbital bleeding (1 and 4 h p.i.; groups Aa, Ba, Ca,
and Ab, Bb, Cb, respectively) 16 2018 Apr. 18 Retro-orbital
bleeding (24 h p.i.; groups Aa, Ba, Ca) 18 2018 Apr. 20
Retro-orbital bleeding (72 h p.i.; groups Ab, Bb, Cb) 21 2018 Apr.
23 IV injection of CA (group D) 21 2018 Apr. 23 Elution of
.sup.212Pb-DOTAM and filling of syringes 22 2018 Apr. 24 IV
injection of.sup.212Pb-DOTAM ) (groups Aa, Ba, Ca, D 22 2018 Apr.
24 Retro-orbital bleeding (168 h p.i.) and euthanasia (groups Ab,
Bb, Cb) 22 2018 Apr. 24 Euthanasia and tissue harvest, incl.
retro-orbital bleeding (6 h p.i.) + gamma counting (groups Aa, Ba,
Ca, D)
Study Groups in Protocol 144
TABLE-US-00032 [0850] P1AD8749 P1AD8592 (VH) (VL) CEA-DOTAM CH1A1A
CH1A1A BsAb PK CA .sup.212Pb BD n Group (.mu.g) (.mu.g) (.mu.g) (h
p.i.) (.mu.g) (.mu.Ci) (h p.i.) (mice) Aa 100 0 0 1, 24, 168 0 10 6
4 Ab 100 0 0 4, 72, 168 0 0 -- 4 Ba 0 100 0 1, 24, 168 0 10 6 4 Bb
0 100 0 4, 72, 168 0 0 -- 4 Ca 100 100 0 1, 24, 168 0 10 6 4 Cb 100
100 0 4, 72, 168 0 0 -- 4 D 0 0 100 -- 25 10 6 4
[0851] Solid xenografts were established in each SCID mouse on
study day 0 by SC injection of 5.times.10.sup.6 cells (passage 26)
in RPMI/Matrigel into the right flank. Fourteen days after tumor
cell injection, mice were sorted into experimental groups with an
average tumor volume of 116 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 22 after inoculation; the average tumor volume was
140 mm.sup.3 on day 21.
[0852] Blood from mice in groups Aa, Ba, and Ca was collected
through retro-orbital bleeding under anesthesia 1 h (right eye), 24
h (left eye), and 168 h (right eye, at termination) after
CEA-split-DOTAM-VH/VL injection. Similarly, samples were taken from
mice in groups Ab, Bb, and Cb 4 h (right eye), 72 h (left eye), and
168 h (right eye, at termination) after CEA-split-DOTAM-VH/VL
injection.
[0853] Mice in groups Aa, Ba, Ca, and D were sacrificed and
necropsied 6 hours after injection of .sup.212Pb-DOTAM, and the
following organs and tissues harvested for measurement of
radioactive content: blood, skin, bladder, stomach, small
intestine, colon, spleen, pancreas, kidneys, liver, lung, heart,
femoral bone, muscle, brain, tail, ears, and tumor.
Results
[0854] The average .sup.212Pb accumulation and clearance in all
collected tissues 6 hours after injection is displayed in FIG. 8.
Pretargeting with either CEA-split-DOTAM-VH or CEA-split-DOTAM-VL
alone resulted in no accumulation of radioactivity in tumors.
Combined, the two complimentary antibodies resulted in a tumor
uptake after 2-step PRIT of 65.+-.12% ID/g, to be compared with
87.+-.15% ID/g for the standard 3-step PRIT regimen. Two-way
analysis of variance (ANOVA) with Tukey's multiple comparisons test
showed that the difference in tumor uptake between the two PRIT
treatments was significant, as was the difference in bladder
(1.+-.2% ID/g and 38.+-.17% ID/g for 2- and 3-step PRIT,
respectively); no other differences in tissue accumulation were
statistically significant using this test (p=0.05).
[0855] The clearance of IV injected CEA-split-DOTAM-VH/VL
constructs as analyzed by an enzyme-linked immunosorbent assay
(ELISA) is shown in FIG. 9.
Adverse Events and Toxicity
[0856] There were no adverse events or toxicity associated with
this study.
Conclusion
[0857] The results of the study demonstrated proof-of-concept of
CA-independent 2-step pretargeting using complimentary
CEA-split-DOTAM-VH/VL antibodies. High and specific tumor uptake of
.sup.212Pb-DOTAM was achieved using 2-step PRIT and standard 3-step
PRIT, with very little accumulation of radioactivity in normal
tissues using the complimentary CEA-split-DOTAM-VH/VL
antibodies.
Example 6c: Protocol 158
[0858] The aim of protocol 158 was to assess the association of
.sup.212Pb-DOTAM to subcutaneous BxPC3 tumors in mice pretargeted
by bi-paratopic (CH1A1A and A5B7) pairs of CEA-split-DOTAM-VH/VL
antibodies for clearing agent-independent 2-step CEA-PRIT. The
tumor uptake was compared with that of standard 3-step
CEA-PRIT.
[0859] Mice carrying subcutaneous BxPC3 tumors were injected with
either [0860] CEA-split-DOTAM-VH/VL antibodies followed 7 days
later by the radiolabeled .sup.212Pb-DOTAM (2-step PRIT), or [0861]
CEA-DOTAM BsAb followed 7 days later by the CA, and finally the
radiolabeled .sup.212Pb-DOTAM 24 hours later (3-step PRIT).
[0862] The in vivo distribution of .sup.212Pb-DOTAM was assessed 6
hours after the radioactive injection. The study outline is shown
in FIG. 10.
Study Design
[0863] The time course and design of protocol 158 is shown in the
tables below.
Time Course of Protocol 158
TABLE-US-00033 [0864] Study day Date Experimental procedure 0 2018
Nov. 26 Preparation of BxPC3 cells and filling of syringes 0 2018
Nov. 26 SC injection of BxPC3 cells 15 2018 Dec. 11 IV injection of
CEA-DOTAM BsAb (group C) 16 2018 Dec. 12 IV injection of
CEA-split-DOTAM-VH/VL BsAbs (groups A, B) 22 2018 Dec.18 IV
injection of CA (group C) 22 2018 Dec.18 Elution of
.sup.212Pb-DOTAM and filling of syringes 23 2018 Dec.19 IV
injection of .sup.212Pb-DOTAM (all) 23 2018 Dec.19 Euthanasia and
tissue harvest, incl retro- orbital bleeding (6 h p.i.) + gamma
counting (all)
Study Groups in Protocol 158
TABLE-US-00034 [0865] P1AD8749 P1AD8592 P1AE4956 P1AE4957 (VH) (VL)
(VH) (VL) CEA-DOTAM CH1A1A CH1A1A A5B7 A5B7 BsAb CA .sup.212Pb n
Group (.mu.g) (.mu.g) (.mu.g) (.mu.g) (.mu.g) (.mu.g) (.mu.Ci)
(mice) A 154* 0 0 100 0 0 10 4 B 0 100 167** 0 0 0 10 4 C 0 0 0 0
100 25 10 4 *P1AD8749 dose adjusted to 154 .mu.g to compensate for
a 35% hole/hole impurity;**P1AD8592 dose adjusted to 167 .mu.g to
compensate for a 40% hole/hole impurity.
[0866] Solid xenografts were established in each SCID mouse on
study day 0 by SC injection of 5.times.10.sup.6 cells (passage 27)
in RPMI/Matrigel into the right flank. Fourteen days after tumor
cell injection, mice were sorted into experimental groups with an
average tumor volume of 177 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 20 after inoculation; the average tumor volume was
243 mm.sup.3 on day 21.
[0867] Mice in all groups were sacrificed and necropsied 6 hours
after injection of .sup.212Pb-DOTAM, and the following organs and
tissues harvested for measurement of radioactive content: blood,
skin, bladder, stomach, small intestine, colon, spleen, pancreas,
kidneys, liver, lung, heart, femoral bone, muscle, brain, tail, and
tumor.
Results
[0868] The average .sup.212Pb distribution in all collected tissues
6 hours after injection is shown in FIG. 11. Two-way ANOVA with
Tukey's multiple comparisons test showed that there was no
significant difference in normal tissue uptake of .sup.212Pb
between the three treatments, except for bladder, where both
bi-paratopic CEA-split-DOTAM-VH/VL pairs yielded lower accumulation
than the standard 3-step PRIT. The kidney uptake was 3-4% ID/g for
all three treatments. Either bi-paratopic combination resulted in
tumor accumulation of approximately 56% ID/g, compared with 67%
ID/g for 3-step PRIT; the difference between 2- and 3-step PRIT was
statistically significant (p<0.0001).
Adverse Events and Toxicity
[0869] There were no adverse events or toxicity associated with
this study.
Conclusion
[0870] This study assessed the association of .sup.212Pb-DOTAM to
SC BxPC3 tumors in mice pretargeted by bi-paratopic pairs of
CEA-split-DOTAM-VH/VL antibodies for CA-independent 2-step
CEA-PRIT, compared with standard 3-step PRIT. The distribution of
.sup.212Pb 6 hours after injection was comparable for 2- and 3-step
PRIT, with high accumulation in tumor and very little radioactivity
in healthy tissues. This demonstrated proof of concept of
bi-paratopic pretargeting of CEA-expressing tumors for 2-step
CEA-PRIT using CEA-split-DOTAM-VH/VL antibodies.
Example 6d: Protocol 160
[0871] The aim of protocol 160 was to compare the therapeutic
efficacy after 3 cycles of CA-independent 2-step CEA-PRIT using
complimentary CEA-split-DOTAM-VH/VL antibodies, with that of
standard 3-step CEA-PRIT in mice bearing SC BxPC3 tumors. A
comparison was also made with 1-step CEA-RIT, using BsAbs that were
pre-incubated with .sup.212Pb-DOTAM before injection.
[0872] Mice carrying SC BxPC3 tumors were injected with either
[0873] CEA-DOTAM BsAb followed 7 days later by the CA, and finally
the radiolabeled .sup.212Pb-DOTAM 24 hours later (3-step PRIT),
[0874] CEA-split-DOTAM-VH/VL antibodies followed 7 days later by
the radiolabeled .sup.212Pb-DOTAM (2-step PRIT), or [0875]
.sup.212Pb-DOTAM-CEA-DOTAM BsAb (pre-incubated; 1-step RIT).
[0876] The therapy was administered in 3 repeated cycles of 20
.mu.Ci of .sup.212Pb-DOTAM, also including comparison with a
non-CEA binding control antibody (DIG-DOTAM), and no treatment
(vehicle). Dedicated mice were sacrificed for biodistribution
purposes to confirm .sup.212Pb-DOTAM targeting and clearance at
each treatment cycle. The treatment efficacy was assessed in terms
of TGI and TR, and the mice were carefully monitored for the
duration of the study to assess the tolerability of the treatment.
The study outline is shown in FIG. 12.
[0877] The time course and design of protocol 160 are shown in the
tables below.
Time Course of Protocol 160
TABLE-US-00035 [0878] Study day Date Experimental procedure 0 2019
Sep. 29 Preparation of BxPC3 cells and filling of syringes 0 2019
Sep. 29 SC injection of BxPC3 cells 15 2019 Sep. 13 IP injection of
BsAb or histidine buffer (groups A, B, C, F, G, H, I) 16 2019
Feb.14 IP injection of CEA-split-DOTAM-VH/VL or histidine buffer
(groups D, J, K, L) 22 2019 Feb.20 IP injection of CA or PBS )
(groups A, B, C, F, G, H, I 23 2019 Feb.21 Elution of
.sup.212Pb-DOTAM and filling of syringes 23 2019 Feb.21 IV
injection of .sup.212Pb-DOTAM-CEA- DOTAM or histidine buffer
(groups E, M) 23 2019 Feb.21 IV injection of .sup.212Pb-DOTAM or
0.9% NaCl (groups B, C, D, F, G, H, I, J, K, L) 24 2019 Feb.22
Euthanasia and tissue harvest (24 h p.i.) + gamma counting (groups
F, G, J, M) 29 2019 Feb.27 IP injection of PRIT BsAb or PBS (groups
A, B, C, H, I) 30 2019 Feb.28 IP injection of CEA-split-DOTAM-VH/VL
or histidine buffer (groups D, K, L) 36 2019 Mar. 06 IP injection
of CA or PBS (groups A, B, C, H, I) 37 2019 Mar. 07 Elution of
.sup.212Pb-DOTAM and filling of syringes 37 2019 Mar. 07 IV
injection of .sup.212Pb-DOTAM or 0.9% NaC1 (groups B, C, D, H, I,
K, L) 38 2019 Mar. 08 Euthanasia and tissue harvest (24 h p.i.) +
gamma counting (groups H, K) 43 2019 Mar. 13 IP injection of PRIT
BsAb or PBS (groups A, B, C, I) 44 2019 Mar. 14 IP injection of
CEA-split-DOTAM-VH/VL or histidine buffer (groups D, L) 50 2019
Mar. 20 IP injection of CA or PBS (groups A, B, C, I) 51 2019 Mar.
21 Elution of Pb-DOTAM and filling of syringes 51 2019 Mar. 21 IV
injection of .sup.212Pb-DOTAM or 0.9% NaC1 (groups B, C, D, I, L)
52 2019 Mar. 22 Euthanasia and tissue harvest (24 h p.i.) + gamma
counting (groups I, L)
Study Groups in Protocol 160
TABLE-US-00036 [0879] BsAb CA .sup.212Pb- per per DOTAM cycle cycle
per cycle Cycles n Group BsAb (.mu.g) (.mu.g) (.mu.Ci) (#) (mice) A
-- 0 0 0 3 10 B DIG-DOTAM 100 25 20 3 10 C CEA-DOTAM 100 25 20 3 10
D CEA-split- 154* + 0 20 3 10 DOTAM 100** E .sup.212Pb-DOTAM- 100 0
20 1** 10 CEA-DOTAM F DIG-DOTAM 100 25 20 1 3 G CEA-DOTAM 100 25 20
1 3 H CEA-DOTAM 100 25 20 2 3 I CEA-DOTAM 100 25 20 3 3 J
CEA-split- 154* + 0 20 1 3 DOTAM 100** K CEA-split- 154* + 0 20 2 3
DOTAM 100** L CEA-split- 154* + 0 20 3 3 DOTAM 100** M
.sup.212Pb-DOTAM- 100 0 20 1 3 CEA-DOTAM *P1AD8749: dose adjusted
to 154 .mu.g to compensate for a 35% hole/hole impurity in the
stock solution; **P1AD8592; ***Adjusted from 3 cycles to 1 cycle
due to acute radiation-induced toxicity at the first treatment
cycle.
[0880] Solid xenografts were established in SCID mice on study day
0 by SC injection of 5.times.10.sup.6 cells (passage 24) in
RPMI/Matrigel into the right flank. Fifteen days after tumor cell
injection, mice were sorted into experimental groups with an
average tumor volume of 122 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 23 after inoculation; the average tumor volume was
155 mm.sup.3 on day 22.
[0881] The CEA-DOTAM and DIG-DOTAM antibodies were diluted in
vehicle buffer to a final concentration of 100 .mu.g per 200 .mu.L
for IP administration according to the table above (Study groups in
protocol 160). The CEA-split-DOTAM-VH/VL antibodies were mixed
together into one single injection solution for IP administration,
containing 100 .mu.g of each construct per 200 .mu.L. For P1AD8749,
the dosing was adjusted to 154 .mu.g to compensate for a 35%
hole/hole impurity in the stock solution (the side of the molecule
that does not carry the VH/VL). The Ca-DOTAM-dextran-500 CA was
administered IP (25 .mu.g per 200 .mu.L of PBS) 7 days after the
BsAb injection, followed 24 hours later by .sup.212Pb DOTAM
(R07205834) according to the experimental schedule in FIG. 12.
PRIT-treated mice (2- and 3-step) were injected IV with 100 .mu.L
of the Ca-quenched .sup.212Pb-DOTAM solution (20 .mu.Ci in 100
.mu.L 0.9% NaCl).
[0882] Mice treated with 1-step RIT received only one injection:
pre-bound .sup.212Pb-DOTAM-CEA-DOTAM (20 .mu.C.sub.1/20 .mu.g BsAb
in 100 .mu.L 0.9% NaCl for IV injection). The direct-labeled
antibody was prepared by incubating the .sup.212Pb-DOTAM with the
CEA-DOTAM BsAb for 10 minutes at 37.degree. C.
[0883] The following organs and tissues were harvested from mice in
groups A-E at the time of euthanasia: serum, liver, spleen,
kidneys, pancreas, and tumor. Before euthanasia, the live mouse was
anesthetized for retro-orbital blood collection. The collected
blood samples were centrifuged at 10 000 ref during 5 minutes and
the resulting serum fractions isolated, frozen, and stored at
-20.degree. C. The excised tissues were immediately put in 10%
neutral buffered formalin (4.degree. C.) and then transferred to
1.times.PBS (4.degree. C.) after 24 hours. The formalin-fixed
samples were shipped to Roche Pharma Research and Early
Development, Roche Innovation Center Basel, for further processing
and analysis.
[0884] Mice in groups F, G, J, and M were sacrificed and necropsied
24 hours after their first and only injection of .sup.212Pb-DOTAM
or .sup.212Pb-DOTAM-BsAb; groups H and K were sacrificed and
necropsied 24 hours after their second .sup.212Pb-DOTAM injection;
groups I and L were sacrificed and necropsied 24 hours after their
third .sup.212Pb-DOTAM injection. Blood was collected at the time
of euthanasia from the venous sinus using retro-orbital bleeding on
anesthetized mice, before termination through cervical dislocation.
The following organs and tissues were also harvested for
biodistribution purposes: bladder, spleen, kidneys, liver, lung,
muscle, tail, skin, and tumor.
Results
[0885] The average .sup.212Pb accumulation and clearance in all
collected tissues 24 hours after injection is shown for each
therapy and treatment cycle in FIG. 13. The negative control
resulted in no uptake (0.4% ID/g) in tumor. Two-way analysis of
variance (ANOVA) with Tukey's multiple comparisons test showed that
the distributions were not significantly different at any cycle for
the 2-step and 3-step PRIT; however, the differences were at all
cycles statistically significant compared with the negative control
and the 1-step RIT (p<0.05). The tumor uptake was 25-45% ID/g
for 3-step PRIT and 25-30% ID/g for 2-step PRIT, without any
statistically significant difference between either treatment or
cycle. For 1-step RIT, the tumor uptake was 99% at the one and only
treatment cycle. The uptake in normal tissues was very low for both
PRIT regimens, but significantly higher in all organs and tissues
after 1-step RIT, due to the much longer circulating time of the
pre-incubated antibody compared with the small, radiolabeled DOTAM
chelate.
[0886] The average tumor development and the individual tumor
growth curves are shown in FIG. 14 and FIG. 15, respectively.
Tumors in the non-treated vehicle group and the DIG-DOTAM group
grew steadily, albeit with slightly lower doubling rate in the
latter after the third treatment. In contrast, tumors in the PRIT
and RIT groups decreased in size after the first treatment cycle,
and maintained tumor control until approximately 10 weeks after
inoculation, when the tumors started to increase in size. The
2-step and 3-step PRIT treatments resulted in near identical tumor
control. No tumors regressed completely.
[0887] On study day 83, the last day on which all treatment groups
could be analyzed based on means, the TGI was 91.7% and 88.4% for
PRIT using CEA-DOTAM (3-step) and CEA-split-DOTAM-VH/VL (2-step),
respectively, compared with the vehicle control. The corresponding
number for 1-step RIT was 72.6%, whereas the TGI was -59.7% for the
non-specific DIG-DOTAM control. On the same day, the TR based on
means was -1.9 for 3-step CEA-DOTAM PRIT, -2.9 for 2-step
CEA-split-DOTAM-VH/VL PRIT, -4.7 for 1-step RIT, -28.8 for
DIG-DOTAM PRIT, and -39.3 for the vehicle control.
[0888] Due to the adverse events described below, survival analysis
was not considered statistically relevant.
Adverse Events and Toxicity
[0889] The BW development in all therapy groups is shown in FIG.
16. The multiple cycles of 2- and 3-step PRIT with 20 .mu.Ci of
.sup.212Pb-DOTAM were well tolerated, but acute BW loss occurred in
mice receiving 1-step RIT, with 8/10 mice in group E euthanized
after the first RIT cycle (6-11 days after .sup.212Pb irradiation)
due to a drop in BW of 20% or more. The remaining 2 RIT mice were
not given any further .sup.212Pb-DOTAM-CEA-DOTAM injections but
were continuously followed up for tumor growth assessment.
[0890] In addition, a number of mice were sacrificed for ethical
reasons due to declining tumor status, i.e. tumors opening up or
leaking. In the DIG-DOTAM group, 9/10 mice were euthanized before
reaching a tumor volume of 3000 mm.sup.3 for this reason; for the
non-treated vehicle control, the corresponding number was 5/10. The
problem was less pronounced in the PRIT and RIT groups, with 1/10,
2/10, and 2/10 mice euthanized for this reason in the 3-step PRIT,
2-step PRIT, and 1-step RIT groups, respectively. This is reflected
in the individual tumor growth curves in FIG. 15.
[0891] Finally, 1 mouse in group C was euthanized due to a
degrading wound under the anus. All adverse events are listed in
the table below.
Adverse Events in Protocol 160
TABLE-US-00037 [0892] Mice sacrificed Reason for Group (n per
group) Study day termination A: Vehicle 5(10) 53, 71, 71, 73, 75
Declining tumor status B : DIG-DOTAM 9(10) 55, 55, 55, 55, 61,
Declining 73, 73, 73, 74 tumor status C : CEA -DOTAM 1(10) 83 Wound
under the anus C : CEA -DOTAM 1(10) 85 Declining tumor status D :
CEA-split-DOTAM 2(10) 83, 83 Declining tumor status E :
.sup.212Pb-DOTAM-CEA- 8(10) 29, 29, 30, 31, 31, BW loss 20% DOTAM
32, 32, 34 E :.sup.212Pb-DOTAM-CEA- 2(10) 83, 83 Declining DOTAM
tumor status .sup.212Pb irradiation was performed on study day 23
(cycle 1), 37 (cycle 2), and 51 (cycle 3).
Conclusion
[0893] No difference was seen between CEA-PRIT using the 3-step
scheme (CEA-DOTAM BsAb, CA, and .sup.212Pb-DOTAM) and the 2-step
scheme (CEA-split-DOTAM-VH/VL antibodies and .sup.212Pb-DOTAM); the
TGI was significant and near identical for the two treatments, and
3 cycles of 20 .mu.C.sub.1 could be safely administered in both
cases. Contrastingly, 20 .mu.C.sub.1 of .sup.212Pb-DOTAM pre-bound
to CEA-DOTAM before injection (1-step RIT) was not tolerated by a
large majority of the treated mice.
The study thus demonstrated tolerability and therapeutic efficacy
of CA-independent 2-step PRIT using the developed
CEA-split-DOTAM-VH/VL constructs.
Example 7: Protocol 175
[0894] The aim of protocol 175 was to assess the impact of
increased injected pretargeting antibody amount on the subsequent
.sup.212Pb accumulation in tumor and healthy tissues. Two different
doses of CEA-split-DOTAM-VH/VL antibodies were compared: the
standard amount (100 .mu.g) and 2.5 times higher dose (250 .mu.g).
Moreover, a modification was made to the CEA-split-DOTAM-VH
construct to extend its VH to avoid anti-drug antibody (ADA)
formation (this was used together with a previously tested
CEA-split-DOTAM-VL construct). The VH was extended to comprise the
first three amino acids from the antibody CH1 domain: alanine,
serine, and threonine (AST), and the construct hereafter referred
to as CEA-split-DOTAM-VH-AST.
[0895] Antibody P1AD8592 has already been described above, in
example 4. P1AF0171 is the same as P1AD8749 except that the fusion
HC is extended by the residues AST--thus, antibody P1AD0171
consists of the light chain D1AA3384 as described above (SEQ ID NO:
34), the first heavy chain D1AC4022 as described above (SEQ ID NO:
28), and a second heavy chain D1AE3669 as shown below:
TABLE-US-00038 D1AE3669 (HC1cnob <CEA> CH1A1A Dotam-VH-AST)
(SEQ ID NO.: 147) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMG
WINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCAR
WDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNICALGAPIEKTISK
AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGGGGGSGGGGSGGGGSGGGGSVTLKESGPVLVKPTETLTL
TCTVSGFSLSTYSMSWIRQPPGICALEWLGFIGSRGDTYYASWAKGRLT
ISKDTSKSQVVLTMTNMDPVDTATYYCARERDPYGGGAYPPHLWGRGTL VTVSSAST
Mice carrying SC BxPC3 tumors were injected with either [0896]
1.times. the standard dose of CEA-split-DOTAM-VH/VL BsAb followed 7
days later by the radiolabeled .sup.212Pb-DOTAM, or [0897]
2.5.times. the standard dose of CEA-split-DOTAM-VH/VL BsAb followed
7 days later by the radiolabeled .sup.212Pb-DOTAM. The in vivo
distribution of .sup.212Pb-DOTAM was assessed 24 hours after the
radioactive injection. The study outline is shown in FIG. 17.
Study Design
[0898] The time course and design of protocol 175 are shown
below.
Time Course of Protocol 175
TABLE-US-00039 [0899] Study day Experimental procedure 0
Preparation of BxPC3 cells and filling of syringes 0 SC injection
of BxPC3 cells 22 IP* injection of CEA-split-DOTAM-VH/VL BsAbs 29
Elution of .sup.212Pb-DOTAM and filling of syringes 29 IV injection
of .sup.212Pb-DOTAM 30 Euthanasia and tissue harvest (24 h p.i.) +
gamma counting *IP injection required due to low compound
concentration (200 .mu.L., per construct = 400 .mu.L in total)
Study Groups in Protocol 175
TABLE-US-00040 [0900] P1AF0171 P1AD85 (VH-AST) 92 (VL) .sup.212Pb
BD n Group (.mu.g) (.mu.g) (.mu.Ci) (h p.i.) (mice) A 143* 100 10
24 4 B 357* 250 10 24 4 *P1AF0171 dose adjusted and 143 to 357
.mu.g to compensate for a ~30% hole/hole impurity.
Solid xenografts were established in each SCID mouse on study day 0
by SC injection of 5.times.10.sup.6 cells (passage 24) in
RPMI/Matrigel into the right flank. Twenty-one days after tumor
cell injection, mice were sorted into experimental groups with an
average tumor volume of 310 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 29 after inoculation; the average tumor volume was
462 mm.sup.3 on day 30. All mice were sacrificed and necropsied 24
hours after injection of .sup.212Pb-DOTAM, and the following organs
and tissues harvested for measurement of radioactive content:
blood, skin, spleen, pancreas, kidneys, liver, muscle, tail, and
tumor.
Results
[0901] The average .sup.212Pb distribution in all collected tissues
24 hours after injection is shown in FIG. 18. There was no
significant difference in tumor or normal tissue uptake of
.sup.212Pb between the two dose levels. The tumor accumulation was
30-31% ID/g for both treatment groups, with a kidney uptake of
<2% ID/g at this time point. One mouse had .about.1% ID/g in the
tail due to .sup.212Pb-DOTAM injection issues, but no other
collected healthy tissues showed any appreciable .sup.212Pb
accumulation.
Adverse Events and Toxicity
[0902] There were no adverse events or toxicity associated with
this study.
Conclusion
[0903] Increasing the dose of the pretargeting
CEA-split-DOTAM-VH/VL antibodies by 2.5-fold did not improve the
tumor accumulation of subsequently administered .sup.212Pb-DOTAM in
this in vivo model. However, it also did not increase the
accumulation of radioactivity in normal tissues, highlighting the
strong specificity achieved using this 2-step pretargeting regimen.
Finally, the results verified the function of the extended-VH
CEA-split-DOTAM-VH-AST construct.
Example 8: Protocol 185
[0904] The aim of protocol 185 was to assess a
CEA-split-DOTAM-VH/VL targeting the T84.66 epitope. Sequences of
P1AF0709 and P1AF0298 are provided herein. P1AF0709 has a first
heavy chain of D1AE4688 (SEQ ID NO: 83) and a second heavy chain of
D1AA4920 (SEQ ID NO: 84). P1AF0298 has a first heavy chain of
D1AE4687 (SEQ ID NO: 86) and a second heavy chain of D1AE3668 (SEQ
ID NO: 87). Both have the light chain of D1AA4120 (SEQ ID NO: 89).
Mice carrying SC BxPC3 tumors were injected with the standard dose
of CEA-split-DOTAM-VH/VL BsAb (100 .mu.g per antibody) followed 6
days later by the radiolabeled .sup.212Pb-DOTAM. The in vivo
distribution of .sup.212Pb-DOTAM was assessed 6 hours after the
radioactive injection. The study outline is shown in FIG. 19.
Study Design
[0905] The time course and design of protocol 185 is shown
below.
Time Course of Protocol 185
TABLE-US-00041 [0906] Study day Date Experimental procedure 0 2020
Mar. 04 Preparation of BxPC3 cells and filling of syringes 0 2020
Mar. 04 SC injection of BxPC3 cells 22 2020 Mar. 26 IV injection of
CEA-split-DOTAM-VH/VL BsAbs 27 2020 Mar. 31 Elution of
.sup.212Pb-DOTAM and filling of syringes 28 2020 Apr. 01 IV
injection of .sup.212Pb-DOTAM 28 2020 Apr. 01 Euthanasia and tissue
harvest (6 h p.i.) + gamma counting
Study Groups in Protocol 185
TABLE-US-00042 [0907] P1AF0298 P1AF0709 P1AF0171 P1AD8592 T84.66
T84.66 CH1A1A CH1A1A (VH-AST) (VL) (VH-AST) (VL) .sup.212Pb BD n
Group (.mu.g) (.mu.g) (.mu.g) (.mu.g) (.mu.Ci) (h p.i.) (mice) A
100 100 0 0 10 6 5 B 0 0 143* 100 10 6 5 *P1AF0171 dose adjusted to
143 .mu.g to compensate for a ~30% hole/hole impurity.
Solid xenografts were established in each SCID mouse on study day 0
by SC injection of 5.times.10.sup.6 cells (passage 27) in
RPMI/Matrigel into the right flank. Twenty-two days after tumor
cell injection, mice were sorted into experimental groups with an
average tumor volume of 224 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 28 after inoculation, at which point the average
tumor volume had reached 385 mm.sup.3. All mice were sacrificed and
necropsied 6 hours after injection of .sup.212Pb-DOTAM, and the
following organs and tissues harvested for measurement of
radioactive content: blood, skin, spleen, pancreas, kidneys, liver,
muscle, tail, and tumor. Collected tumors were split in two pieces:
one was measured for radioactive content, and the other put in a
cryomold containing Tissue-Tek.RTM. optimum cutting temperature
(OCT) embedding medium, and put on dry ice for rapid freezing.
Frozen samples in OCT were maintained at -80.degree. C. before
cryosectioning, immunofluorescence staining, and analysis using a
Zeiss Axio Scope.A1 modular microscope.
Results
[0908] The average .sup.212Pb distribution in all collected tissues
6 hours after injection is shown in FIG. 20. The tumor accumulation
was 40% ID/g (CH1A1A) or 44% ID/g (T84.66). The only other
appreciable accumulation of radioactivity was found in kidneys:
3-5% ID/g at 6 h p.i. for the two groups. Examples of the
intratumoral distribution of CEA-split-DOTAM-VH/VL pairs targeting
either T84.66 (group A) or CH1A1A (group B) are shown in FIG. 21.
Panels A and C show that the CEA expression is high and homogeneous
in BxPC3 tumors, and panels B and D demonstrate that the antibody
distribution 7 days after injection is distributed similarly.
However, the samples from group A displayed a stronger signal
overall, compared with tumor samples from group B, providing
evidence that T84.66 is a stronger binder than CH1A1A.
Adverse Events and Toxicity
[0909] There were no adverse events or toxicity associated with
this study.
Conclusion
[0910] The results verified the function of CEA-split-DOTAM-VH/VL
constructs targeting the T84.66 epitope of CEA. The resulting
accumulation of 212Pb in pretargeted CEA-expressing tumors was high
and specific, and CEA-split-DOTAM-VH/VL pairs targeting either the
CH1A1A or T84.66 epitope were homogeneously distributed inside the
CEA-expressing tumors.
Example 9: Protocol 189
[0911] The aim of protocol 189 was to assess bi-paratopic
CEA-split-DOTAM-VH/VL antibody pairs targeting T84.66 VH-AST/CH1A1A
VL and T84.66 VL/CH1A1 VH-AST, compared with the positive control
pair targeting CH1A1A VH-AST/VL. This bi-paratopic combination
precludes formation of the full Pb-DOTAM binder on soluble CEA that
only expresses one of the two epitopes (e.g. T84.66), thereby
mitigating potential adverse effects thereof, such as increased
circulating radioactivity and associated radiation-induced
toxicity, and decreased efficacy from competition with off-tumor
targets. Mice carrying SC BxPC3 tumors were injected with the
standard dose of CEA-split-DOTAM-VH/VL BsAb (100 .mu.s per
antibody) followed 7 days later by the radiolabeled
.sup.212Pb-DOTAM. The in vivo distribution of .sup.212Pb-DOTAM was
assessed 6 hours after the radioactive injection. The study outline
is shown in FIG. 22.
Study Design
[0912] The time course and design of protocol 189 is shown
below.
Time Course of Protocol 189
TABLE-US-00043 [0913] Study day Experimental procedure 0
Preparation of BxPC3 cells and filling of syringes 0 SC injection
of BxPC3 cells 15 IV injection of CEA-split-DOTAM-VH/VL BsAbs 21
Elution of .sup.212Pb-DOTAM and filling of syringes 22 IV injection
of .sup.2I2Pb-DOTAM _ 22 Euthanasia and tissue harvest (6 h p.i.) +
gamma counting
Study Groups in Protocol 189
TABLE-US-00044 [0914] P1AF0298 P1AF0709 P1AF0171 P1AD8592 T84.66
T84.66 CH1A1A CH1A1A (VH-AST) (VL) (VH-AST) (VL) .sup.212Pb BD n
Group (.mu.g) (.mu.g) (.mu.g) (.mu.g) (.mu.Ci) (h p.i.) (mice) A
100 0 0 100 10 6 5 B 0 100 143* 0 10 6 5 C 0 0 143* 100 10 6 3
*P1AF0171 dose adjusted to 143 .mu.g to compensate for a ~30%
hole/hole impurity.
Solid xenografts were established in each SCID mouse on study day 0
by SC injection of 5.times.10.sup.6 cells (passage 31) in
RPMI/Matrigel into the right flank. Fourteen days after tumor cell
injection, mice were sorted into experimental groups with an
average tumor volume of 343 mm.sup.3. The .sup.212Pb-DOTAM was
injected on day 22 after inoculation; the average tumor volume had
reached 557 mm.sup.3 on day 21. All mice were sacrificed and
necropsied 6 hours after injection of .sup.212Pb-DOTAM, and the
following organs and tissues harvested for measurement of
radioactive content: blood, skin, spleen, pancreas, kidneys, liver,
muscle, tail, and tumor.
Results
[0915] The average .sup.212Pb distribution in all collected tissues
6 hours after injection is shown in FIG. 23. The tumor accumulation
of the bi-paratopic variations was 71% ID/g and 46% ID/g for T84.66
VH-AST+CH VL and T84.66 VL+CH VH-AST, respectively. The positive CH
control resulted in 37% ID/g. Two-way ANOVA with Tukey's multiple
comparisons test showed that all three groups were significantly
different from each other in terms of tumor uptake (p<0.0001 for
T84.66 VH-AST+CH1A1A VL versus the two other groups; p=0.0020 for
T84.66 VL+CH VH-AST versus CH1A1A only). No other organs showed
statistically significant differences between groups, although a
slightly higher retention in blood was indicated for the T84.66
VH-AST+CH1A1A VL combination compared with the two other groups: 2%
ID/g compared with <1% ID/g. The kidney uptake was similarly
slightly higher, although not statistically significantly so: 4.5%
ID/g for T84.66 VH-AST+CH1A1A compared with 3% ID/g for the other
two.
Adverse Events and Toxicity
[0916] There were no adverse events or toxicity associated with
this study. However, the BxPC3 tumor growth was significantly
faster, and with greater variability, in this study compared with
the standard growth rate. On necropsy, it was concluded that the
big tumors (a majority) were filled with liquid, which was emptied
when tumors were cut in half before radioactive measurement; this
liquid likely caused the accelerated growth rate, but did not
affect the % IA/g to any great extent as the tumors were weighed
and measured after being opened.
Conclusion
[0917] The results verified the function of bi-paratopic targeting
of the T84.66 and CH1A1A epitopes of CEA using the tested
CEA-split-DOTAM-VH/VL constructs and demonstrated surprisingly high
efficacy for this combination as compared to the positive CH1A1A
control. The resulting accumulation of .sup.212Pb in pretargeted
CEA-expressing tumors was high and specific, with indications of a
particular advantage for the T84.66 VH-AST+CH1A1A VL pair.
Example 10
[0918] These examples investigate recruitment of Pb-DOTA to cells
by split antibodies as described herein. P1AF0712 has a first heavy
chain of SEQ ID NO:97, a second heavy chain of SEQ ID NO: 98 and a
light chain of SEQ ID NO: 103. P1AF0713 has a first heavy chain of
SEQ ID NO: 100, a second heavy chain of SEQ ID NO: 101 and a light
chain of SEQ ID NO: 103. MKN-45 cells were detached from the
culture bottle using Trypsin and were counted using a Casy cell
counter. After pelleting at 4.degree. C., 300 g the cells were
resuspended in FACS Buffer (2.5% FCS in PBS), adjusted to 2.0E+06
cells/mL dispensed to 96-well PP V-bottom-Platte (25
.mu.L/well=5.0E+04 Zellen/well).
FACS Staining Using DOTA-FITC
[0919] The CEA specific SPLIT antibodies (P1AF0712 or P1AF0713
respectively) were adjusted to 40 .mu.g/mL in FACS buffer,
resulting in a final concentration of 10 .mu.g/mL. Both antibodies
were added to the cells either combined or separated and followed
by buffer and incubated at 4.degree. C. for 1 h. Subsequently,
Pb-DOTA labeled with FITC was added to the cells in equimolar ratio
to the antibodies and incubated for 1 h at 4.degree. C. The cells
were then washed twice in FACS buffer and resuspended in 70
.mu.l/well FACS buffer for measurement using a FACS Canto (BD,
Pharmingen). It was shown (FIG. 24) that neither of the SPLIT
halves was giving rise to a fluorescence signal, indicating a lack
of Pb-DOTA binding capability. Only a combination of both SPLIT
halves was able to recruit Pb-DOTAM-FITC to the target cells (FIG.
24). FACS Staining Using <huIgG(H+L)A488> The CEA specific
SPLIT antibodies (P1AF0712 or P1AF0713 respectively) were adjusted
to 40 .mu.g/mL in FACS buffer, resulting in a final concentration
of 10 .mu.g/mL. Both antibodies were added to the cells either
separated followed by buffer or combined and incubated at 4.degree.
C. for 1 h. The cells were then washed twice in FACS buffer. After
washing, the cells were resuspended in 50 .mu.L FACS-buffer
containing secondary antibody (<huIgG(H+L)>-Alexa488, c=10
.mu.g/mL) and incubated 1 h at 4.degree. C. The cells were then
washed twice in FACS buffer and resuspended in 70 .mu.l/well FACS
buffer for measurement using a FACS Canto (BD, Phaimingen). EC50
for both SPLIT antibodies was comparable, indicating CEA specific
cell binding of both SPLIT antibodies. Due to the higher amount of
antibody in the mixture, a lower EC50 was obtained under these
circumstances, as shown in the table below.
EC50 Determination of SPLIT Antibodies
TABLE-US-00045 [0920] EC50 .mu.g/ml absolute <hu>A488
P1AF0712+PBS 2.7 P1AF0713+PBS 2.3 P1AF0712+P1AF0713 0.9 hu ISO +PBS
DOTA-FITC P1AF0712+PBS na P1AF0713+PBS na P1AF0712+P1AF0713 2.4 hu
ISO +PBS
EC50 was determined for the SPLIT antibodies using either secondary
antibody based detection (<hu>488, top panel) or Pb-DOTA-FITC
(DOTA-FITC, bottom panel)
Example 11: Biacore Binding Experiments
[0921] This example tests binding of TA-split-DOTAM-VH and
TA-split-DOTAM-VL individually to DOTAM, as compared to the
reference antibody CEA-DOTAM (R07198427, PRIT-0213). It further
tests binding of DOTAM to the TA-split-DOTAM-VH/VL pairs, as
compared to the reference antibody. The correspondence between the
coding used in these examples and the protein numbers used
elsewhere in this application is shown below. Sequences are also
provided. The reference antibody is coded as "PRIT_RS" in this
example.
TABLE-US-00046 SPR Code LC HC Fusion HC (Prodrug SPR (SEQ (SEQ (SEQ
Target binder A + B) Code DOTAM Protein ID NO) ID NO) ID NO)
<CEA> CH1A1A P1_AB P1_A VL P1AD8592 34 30 33 P1 _B VH
P1AD8749 34 28 32 <CEA> CH1A1A P2_AB P2_A VL P1AD8592 34 30
33 P2_B VH P 1AF0171 34 28 147 <CEA> T84.66 P3_AB P3_A VL
P1AF0709 89 83 84 P3_B VH P1AF0298 89 86 89 <CEA> 28A9 P4_AB
P4_A VL P1AF0710 96 90 91 P4 _B VH P1AF0711 96 93 94 <CEA>
P5_AB P5_A VL P1AE4957 58 55 56 A5H1EL1(G54A) P5_B VH P1AE4956 54
51 52 <CEA> CH1A1A P6_AB P6_A VL P1AF0712 103 97 98 P6_B VH
P1AF0713 103 100 101 <GPRC5D> P7_AB P7_A VL P1AF8284 107 104
105 P7_B VH P1AF8285 107 104 106
For these experiments, the PRIT SPLIT antibodies were purified by a
first step of MabSelect Sure (Affinity Chromatography) and a second
step of ion exchange chromatography (e.g. POROS XS), and then
polished by Superdex 200 (Size Exclusion Chromatography). The
experiments were performed with Biacore T200 at 25.degree. C.
measuring temperature. All Biacore T200 experiments were carried
out in HBS-P+(GE Healthcare, Br-1008-27) pH 7,4 running buffer. Two
experiments were performed for each test antibody/antibody pair,
using different DOTAM fractions. 1. In a first experiment, the
binding of individual TA-split-DOTAM-VH and TA-split-DOTAM-VL
antibodies to biotinylated DOTAM captured on a chip was assessed,
relative to the reference antibody. DOTAM (120 nM solution in
HBS-P+) was captured in high density on CAP Chip Surface (10
.mu.l/min, 60 Sec). Then the 600 nM solutions in HBS-P+ of
Prodrug_A or Prodrug_B were injected over the DOTAM surface (10
.mu.l/min, 90 sec). The dissociation was monitored for 240 sec at a
flow rate of 10 .mu.l/min. The relative maximum response
determination was evaluated using T200 evaluation software. The
results are shown in FIG. 26. None of the individual antibodies
showed binding to the captured DOTAM. 2. In a second experiment,
individual TA-split-DOTAM-VH and TA-split-DOTAM-VL antibodies were
first captured in a chip using an immobilized anti-hFab, and then
binding of a DOTAM-monoStreptavidin complex (DOTAM+monoSteptavidin
coupling 600 nM, 1:1 mol, 1 h at RT) was assessed. The 600 nM
solution in HBS-P+ of Prodrug_A or Prodrug B was injected over the
anti hFab (GE Healthcare, BR-1008-27) CM5 Chip surface (10
.mu.l/min, 120 sec). After the high density capturing of Prodrug A
or B solution the DOTAM-monoStreptavidin complex was injected (20
.mu.l/min, 90 sec). The dissociation was monitored for 180 sec at a
flow rate of 20 .mu.l/min. For new cycle the surface was
regenerated by using of Glycin 2.1 and 75 sec regeneration time
with 10 .mu.l/min. The relative maximum response determination was
evaluated using T200 evaluation software. The results are shown in
FIG. 27. Low percentage max. responses (as marked with * in the
figure) are believed to be "traces" or unspecific interactions with
DOTAM-SA, and reflect a need to optimize the assay. 3. In a third
experiment, binding of the TA-split-DOTAM-VH/VL pairs to DOTAM is
assessed, as compared to the reference antibody. Antibodies were
first captured in a chip using an immobilized anti-hFab, and then
binding of a DOTAM-monoStreptavidin complex (DOTAM+monoSteptavidin
coupling 600 nM, 1:1 mol, 1 h at RT) was assessed. The 300 nM
solution in HBS-P+ of Prodrug_A and Prodrug B was injected over the
anti hFab (GE Healthcare, BR-1008-27) CM5 Chip surface (10
.mu.l/min, 120 sec). After the high density capturing of Prodrug A
and B solution the DOTAM-monoStreptavidin complex was injected (20
.mu.l/min, 90 sec). The dissociation was monitored for 180 sec at a
flow rate of 20 .mu.l/min. For new cycle the surface was
regenerated by using of Glycin 2.1 and 75 sec regeneration time
with 10 .mu.l/min. The relative maximum response determination was
evaluated using T200 evaluation software. The results are shown in
FIG. 28. All TA-split-DOTAM-VH/VL pairs showed a significant amount
of binding for DOTAM, except for the P6_AB (P1AF0712/P1AF0713)
pair, which are DOTA binders. Similar results showing a significant
amount of DOTAM binding for the TA-split-DOTAM-VH/VL pair but not
for the individual members of the pair have also been obtained for
the FAP-binders P1AF8286 and P1AF8287. P1AF8286 is composed of a
first heavy chain of SEQ ID NO: 108, a second heavy chain of SEQ ID
NO: 109 and a light chain of SEQ ID NO: 111, and P1AF8287 is
composed of a first heavy chain of SEQ ID NO: 108, a second heavy
chain of SEQ ID NO: 110 and a light chain of SEQ ID NO: 111.
However, this assay still needs to be optimised. Although the
foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
the descriptions and examples should not be construed as limiting
the scope of the invention. The disclosures of all patent and
scientific literature cited herein are expressly incorporated in
their entirety by reference.
Sequence CWU 1
1
180110PRTArtificial Sequenceheavy chain CDR1, <Pb-Dotam> 1Gly
Phe Ser Leu Ser Thr Tyr Ser Met Ser1 5 10216PRTArtificial
Sequenceheavy chain CDR2 <Pb-Dotam> 2Phe Ile Gly Ser Arg Gly
Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly1 5 10 15314PRTArtificial
Sequenceheavy chain CDR3 <Pb-Dotam> 3Glu Arg Asp Pro Tyr Gly
Gly Gly Ala Tyr Pro Pro His Leu1 5 10413PRTArtificial Sequencelight
chain CDR1, <Pb-Dotam> 4Gln Ser Ser His Ser Val Tyr Ser Asp
Asn Asp Leu Ala1 5 1057PRTArtificial Sequencelight chain CDR2
<Pb-Dotam> 5Gln Ala Ser Lys Leu Ala Ser1 5612PRTArtificial
Sequencelight chain CDR3 <Pb-Dotam> 6Leu Gly Gly Tyr Asp Asp
Glu Ser Asp Thr Tyr Gly1 5 107121PRTArtificial Sequenceheavy chain
variable domain <Pb-Dotam> PRIT-0213 7Val Thr Leu Lys Glu Ser
Gly Pro Val Leu Val Lys Pro Thr Glu Thr1 5 10 15Leu Thr Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 35 40 45Phe Ile Gly
Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Leu
Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr65 70 75
80Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
85 90 95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
1208111PRTArtificial Sequencelight chain variable domain
<Pb-Dotam> PRIT-0213 8Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly Asp1 5 10 15Arg Val Thr Ile Thr Cys Gln Ser Ser
His Ser Val Tyr Ser Asp Asn 20 25 30Asp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gln Ala Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu 85 90 95Ser Asp Thr
Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
1109121PRTArtificial Sequenceheavy chain variable domain
<Pb-Dotam> PRIT-0214 9Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu Thr1 5 10 15Leu Ser Leu Thr Cys Ala Val Tyr Gly
Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45Phe Ile Gly Ser Arg Gly Asp
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Val Thr Ile Ser Arg
Asp Thr Ser Lys Asn Gln Val Ser Leu Lys65 70 75 80Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95Glu Arg Asp
Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly 100 105 110Arg
Gly Thr Leu Val Thr Val Ser Ser 115 12010111PRTArtificial
Sequencelight chain variable domain <Pb-Dotam> PRIT-0214
10Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp1
5 10 15Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp
Asn 20 25 30Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gly Gly Tyr Asp Asp Glu 85 90 95Ser Asp Thr Tyr Gly Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 1101110PRTArtificial Sequenceheavy
chain CDR1 <CEA> T84.66 11Gly Phe Asn Ile Lys Asp Thr Tyr Met
His1 5 101217PRTArtificial Sequenceheavy chain CDR2 <CEA>
T84.66 12Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys
Phe Gln1 5 10 15Gly1312PRTArtificial Sequenceheavy chain CDR3
<CEA> T84.66 13Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala
Tyr1 5 101415PRTArtificial Sequencelight chain CDR1 <CEA>
T84.66 14Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu
His1 5 10 15157PRTArtificial Sequencelight chain CDR2 <CEA>
T84.66 15Arg Ala Ser Asn Arg Ala Thr1 5169PRTArtificial
Sequencelight chain CDR3 <CEA> T84.66 16Gln Gln Thr Asn Glu
Asp Pro Tyr Thr1 517121PRTArtificial Sequenceheavy chain variable
domain <CEA> T84.66 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn
Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe
Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115 12018111PRTArtificial
Sequencelight chain variable domain <CEA> T84.66 18Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25
30Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 1101910PRTArtificial Sequenceheavy chain
CDR1 <CEA> CH1A1A 19Gly Tyr Thr Phe Thr Glu Phe Gly Met Asn1
5 102017PRTArtificial Sequenceheavy chain CDR2 <CEA> CH1A1A
20Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys1
5 10 15Gly2112PRTArtificial Sequenceheavy chain CDR3 <CEA>
CH1A1A 21Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr1 5
102211PRTArtificial Sequencelight chain CDR1 <CEA> CH1A1A
22Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala1 5 10237PRTArtificial
Sequencelight chain CDR2 <CEA> CH1A1A 23Ser Ala Ser Tyr Arg
Lys Arg1 52410PRTArtificial Sequencelight chain CDR3 <CEA>
CH1A1A 24His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr1 5
1025121PRTArtificial Sequenceheavy chain variable domain
<CEA> CH1A1A 25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly
Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr
Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp
Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 12026108PRTArtificial Sequencelight
chain variable domain <CEA> CH1A1A 26Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu
85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10527450PRTArtificial SequenceHeavy chain <CEA> of P1AD8749
without linker and <DOTAM-VH>' Same Plasmid as SeqID32,
lacking linker and <DOTAM> 27Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr
Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val
Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45028450PRTArtificial SequenceP1AD8749 Heavy chain hole <CEA>
CH1A1A 28Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr
Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr
Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265
270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Ser Cys
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390
395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 435 440 445Pro Gly 45029450PRTArtificial
SequenceHeavy chain <CEA> of P1AD8592 without linker and
<DOTAM-VL>' Same Plasmid as SeqID33, lacking linker and
<DOTAM> 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala
Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala
Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45030450PRTArtificial SequenceP1AD8592 Heavy chain Knob
<CEA>CH1A1A 30Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu
Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe
Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
4503120PRTArtificial SequenceLinker 31Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
2032591PRTArtificial SequenceP1AD8749 heavy chain knob
<CEA>CH1A1A<Dotam-VH> 32Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr
Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val
Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser
Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu465 470
475 480Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly
Phe 485 490 495Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro
Pro Gly Lys 500 505 510Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg
Gly Asp Thr Tyr Tyr 515 520 525Ala Ser Trp Ala Lys Gly Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys 530 535 540Ser Gln Val Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala545 550 555 560Thr Tyr Tyr Cys
Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr 565 570 575Pro Pro
His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 580 585
59033581PRTArtificial SequenceP1AD8592 heavy chain hole
<CEA>CH1A1A<Dotam-VL> 33Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr
Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val
Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser
Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu465 470
475 480Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser
His 485 490 495Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln
Lys Pro Gly 500 505 510Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser
Lys Leu Ala Ser Gly 515 520 525Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu 530 535 540Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu545 550 555 560Gly Gly Tyr Asp
Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr 565 570 575Lys Val
Glu Ile Lys 58034215PRTArtificial SequenceP1AD8749 and P1AD8592
light chain <CEA> CH1A1A 34Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr
Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95Phe
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn
Arg Gly Glu Cys 210 215355PRTArtificial SequenceHeavy chain CDR 1,
<C825> 35Asp Tyr Gly Val His1 53616PRTArtificial
SequenceHeavy chain CDR 2, <C825> 36Val Ile Trp Ser Gly Gly
Gly Thr Ala Tyr Asn Thr Ala Leu Ile Ser1 5 10 153711PRTArtificial
SequenceHeavy chain CDR 3, <C825> 37Arg Gly Ser Tyr Pro Tyr
Asn Tyr Phe Asp Ala1 5 103814PRTArtificial SequenceLight chain CDR
1, <C825> 38Gly Ser Ser Thr Gly Ala Val Thr Ala Ser Asn Tyr
Ala Asn1 5 10397PRTArtificial SequenceLight chain CDR 2,
<C825> 39Gly His Asn Asn Arg Pro Pro1 5409PRTArtificial
SequenceLight chain CDR 3, <C825> 40Ala Leu Trp Tyr Ser Asp
His Trp Val1 541119PRTArtificial SequenceHeavy chain variable
domain <C825> 41His Val Lys Leu Gln Glu Ser Gly Pro Gly Leu
Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Asp Tyr 20 25 30Gly Val His Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Gly
Thr Ala Tyr Asn Thr Ala Leu Ile 50 55 60Ser Arg Leu Asn Ile Tyr Arg
Asp Asn Ser Lys Asn Gln Val Phe Leu65 70 75 80Glu Met Asn Ser Leu
Gln Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95Arg Arg Gly Ser
Tyr Pro Tyr Asn Tyr Phe Asp Ala Trp Gly Gln Gly 100 105 110Thr Thr
Val Thr Val Ser Ser 11542109PRTArtificial SequenceLight chain
variable domain, <C825> 42Gln Ala Val Val Ile Gln Glu Ser Ala
Leu Thr Thr
Pro Pro Gly Glu1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly
Ala Val Thr Ala Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro
Asp His Leu Phe Thr Gly 35 40 45Leu Ile Gly Gly His Asn Asn Arg Pro
Pro Gly Val Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys
Ala Ala Leu Thr Ile Ala Gly Thr65 70 75 80Gln Thr Glu Asp Glu Ala
Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asp 85 90 95His Trp Val Ile Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105435PRTArtificial
Sequenceheavy chain CDR1 <CEA> A5B7 43Asp Tyr Tyr Met Asn1
54419PRTArtificial Sequenceheavy chain CDR2 <CEA> A5B7 44Phe
Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser1 5 10
15Val Lys Gly4510PRTArtificial Sequenceheavy chain CDR3 <CEA>
A5B7 45Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr1 5
104610PRTArtificial Sequencelight chain CDR1 <CEA> A5B7 46Arg
Ala Ser Ser Ser Val Thr Tyr Ile His1 5 10477PRTArtificial
Sequencelight chain CDR2 <CEA> A5B7 47Ala Thr Ser Asn Leu Ala
Ser1 5489PRTArtificial Sequencelight chain CDR3 <CEA> A5B7
48Gln His Trp Ser Ser Lys Pro Pro Thr1 549121PRTArtificial
Sequenceheavy chain variable domain <CEA> A5B7 49Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala
50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn
Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr 85 90 95Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12050106PRTArtificial Sequencelight chain variable domain
<CEA> A5B7 50Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser
Ser Val Thr Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Ser Trp Ile Tyr 35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Ile
Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr
Cys Gln His Trp Ser Ser Lys Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 10551450PRTArtificial SequenceP1AE4956
heavy chain hole <CEA> A5B7 51Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly Asn
Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr65 70 75 80Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95Tyr
Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45052591PRTArtificial SequenceP1AE4956 heavy chain knob <CEA>
A5B7 <Dotam-VH> 52Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly Asn Lys Ala Asn
Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Lys Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95Tyr Cys Thr Arg
Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu465 470 475
480Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
485 490 495Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro
Gly Lys 500 505 510Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly
Asp Thr Tyr Tyr 515 520 525Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile
Ser Lys Asp Thr Ser Lys 530 535 540Ser Gln Val Val Leu Thr Met Thr
Asn Met Asp Pro Val Asp Thr Ala545 550 555 560Thr Tyr Tyr Cys Ala
Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr 565 570 575Pro Pro His
Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 580 585
59053449PRTArtificial SequenceHeavy chain <CEA> of P1AE4956
without linker and DOTAM-VH 53Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly Asn Lys
Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95Tyr Cys
Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro54213PRTArtificial SequenceP1AE4956 light chain <CEA>
A5B7 54Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr
Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser
Trp Ile Tyr 35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln His
Trp Ser Ser Lys Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
21055450PRTArtificial SequenceP1AE4957 heavy chain knob <CEA>
A5B7 55Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr
Asp Tyr 20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr
Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Lys Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Thr Tyr 85 90 95Tyr Cys Thr Arg Asp Arg Gly Leu
Arg Phe Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45056581PRTArtificial SequenceP1AE4957 heavy chain hole <CEA>
A5B7 <Dotam-VL> 56Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly Asn Lys Ala Asn
Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Lys Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95Tyr Cys Thr Arg
Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu465 470 475
480Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His
485 490 495Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly 500 505 510Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys
Leu Ala Ser Gly 515 520 525Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu 530 535 540Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Leu545 550 555 560Gly Gly Tyr Asp Asp
Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr 565 570 575Lys Val Glu
Ile Lys 58057449PRTArtificial SequenceHeavy chain <CEA> of
P1AE4957 without linker and DOTAM-VL 57Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly
Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr65 70 75 80Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90
95Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro58213PRTArtificial SequenceP1AE4957 light chain <CEA>
A5B7 58Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr
Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser
Trp Ile Tyr 35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln His
Trp Ser Ser Lys Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 2105910PRTArtificial
Sequenceheavy chain CDR1 <CEA> 28A9 59Gly Gly Thr Phe Ser Tyr
Tyr Ala Ile Ser1 5 106017PRTArtificial Sequenceheavy chain CDR2
<CEA> 28A9 60Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala
Gln Lys Phe Gln1 5 10 15Gly6111PRTArtificial Sequenceheavy chain
CDR3 <CEA> 28A9 61Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp
Tyr1 5 106211PRTArtificial Sequencelight chain CDR1 <CEA>
28A9 62Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala1 5
10637PRTArtificial Sequencelight chain CDR2 <CEA> 28A9 63Asp
Ala Ser Ser Leu Glu Ser1 5649PRTArtificial Sequencelight chain CDR3
<CEA> 28A9 64Gln Gln Asn Thr Gln Tyr Pro Met Thr1
565120PRTArtificial Sequenceheavy chain variable domain <CEA>
28A9 65Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser
Tyr Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala Asn Tyr
Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser
Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Pro Pro Leu Pro Gly
Ala Gly Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val
Ser Ser 115 12066107PRTArtificial Sequencelight chain variable
domain <CEA> 28A9 66Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asn Thr Gln Tyr Pro Met 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 1056710PRTArtificial
Sequenceheavy chain CDR1 <GPRC5D> 67Gly Phe Thr Phe Ser Lys
Tyr Ala Met Ala1 5 106817PRTArtificial Sequenceheavy chain CDR2
<GPRC5D> 68Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala
Asp Ser Val Lys1 5 10 15Gly698PRTArtificial Sequenceheavy chain
CDR3 <GPRC5D> 69His Thr Gly Asp Tyr Phe Asp Tyr1
57015PRTArtificial Sequencelight chain CDR1 <GPRC5D> 70Arg
Ala Ser Gln Ser Val Ser Ile Ser Gly Ile Asn Leu Met Asn1 5 10
15717PRTArtificial Sequencelight chain CDR2 <GPRC5D> 71His
Ala Ser Ile Leu Ala Ser1 5729PRTArtificial Sequencelight chain CDR3
<GPRC5D> 72Gln Gln Thr Arg Glu Ser Pro Leu Thr1
573117PRTArtificial SequenceHeavy chain variable domain
<GPRC5D> 73Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Lys Tyr 20 25 30Ala Met Ala Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Thr Gly Gly Val Asn
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr His Thr Gly
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met 100 105 110Val Thr Val
Ser Ser 11574111PRTArtificial SequenceLight chain variable domain
<GPRC5D> 74Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ile Ser 20 25 30Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln
Lys Pro Gly Gln Gln Pro 35 40 45Lys Leu Leu Ile Tyr His Ala Ser Ile
Leu Ala Ser Gly Ile Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Arg Leu Glu Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Thr Arg 85 90 95Glu Ser Pro Leu Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
1107510PRTArtificial Sequenceheavy chain CDR1 <FAP> 4B9 75Gly
Phe Thr Phe Ser Ser Tyr Ala Met Ser1 5 107617PRTArtificial
Sequenceheavy chain CDR2 <FAP> 4B9 76Ala Ile Ile Gly Ser Gly
Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly778PRTArtificial
Sequenceheavy chain CDR3 <FAP> 4B9 77Gly Trp Phe Gly Gly Phe
Asn Tyr1 57812PRTArtificial Sequencelight chain CDR1 <FAP>
4B9 78Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr Leu Ala1 5
10797PRTArtificial Sequencelight chain CDR2 <FAP> 4B9 79Val
Gly Ser Arg Arg Ala Thr1 5809PRTArtificial Sequencelight chain CDR3
<FAP> 4B9 80Gln Gln Gly Ile Met Leu Pro Pro Thr1
581117PRTArtificial SequenceHeavy chain variable domain <FAP>
4B9 81Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Trp Phe Gly Gly Phe
Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11582108PRTArtificial SequenceLight chain variable domain
<FAP> 4B9 82Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Thr Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu
35 40 45Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly
Ile Met Leu Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10583450PRTArtificial SequenceP1AF0709 HCknob <CEA>
T84.66 (D1AE4688) 83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn
Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr
Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45084581PRTArtificial SequenceP1AF0709 HChole <CEA> T84.66
Dotam-VL (D1AA4920) 84Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly
Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu465 470 475
480Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His
485 490 495Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly 500 505 510Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys
Leu Ala Ser Gly 515 520 525Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu 530 535 540Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Leu545 550 555 560Gly Gly Tyr Asp Asp
Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr 565 570 575Lys Val Glu
Ile Lys 58085450PRTArtificial SequenceP1AF0709 HChole <CEA>
T84.66 without linker and DOTAM 85Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro
Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45086450PRTArtificial SequencePIAF0298 HCHole <CEA> T84.66
(D1AE4687) 86Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn
Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser
Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr
Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45087594PRTArtificial SequencePIAF0298 HCknob <CEA> T84.66
Dotam-VH-AST (D1AE3668) 87Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn
Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe
Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455
460Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val
Leu465 470 475 480Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe 485 490 495Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile
Arg Gln Pro Pro Gly Lys 500 505 510Ala Leu Glu Trp Leu Gly Phe Ile
Gly Ser Arg Gly Asp Thr Tyr Tyr 515 520 525Ala Ser Trp Ala Lys Gly
Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 530 535 540Ser Gln Val Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala545 550 555 560Thr
Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr 565 570
575Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala
580 585 590Ser Thr88450PRTArtificial SequencePIAF0298 HCknob
<CEA> T84.66 without linker and DOTAM 88Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly 45089218PRTArtificial SequenceP1AF0709 and PIAF0298
light chain (D1AA4120) 89Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Gly Glu Ser Val Asp Ile Phe 20 25 30Gly Val Gly Phe Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95Glu Asp Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21590449PRTArtificial SequenceP1AF710
HCknob <CEA> 28A9 (D1AE4690) 90Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Leu
Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 355 360 365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly91580PRTArtificial SequenceP1AF710 HChole <CEA> 28A9
Dotam-VL (D1AC3172) 91Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Leu Pro Ala Phe Gly
Ala Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Pro
Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460Gly Gly Gly
Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser465 470 475
480Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser
485 490 495Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys 500 505 510Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu
Ala Ser Gly Val 515 520 525Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr 530 535 540Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly545 550 555 560Gly Tyr Asp Asp Glu
Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys 565 570 575Val Glu Ile
Lys 58092449PRTArtificial SequenceP1AF710 HChole <CEA> 28A9
without linker or DOTAM 92Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Leu Pro Ala Phe
Gly Ala Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu
Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360
365Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly93449PRTArtificial SequenceP1AF711 HChole <CEA> 28A9
(D1AE4689) 93Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Leu Pro Ala Phe Gly Ala Ala
Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr
Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Cys 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445Gly94593PRTArtificial SequenceP1AF711 1 HCknob
<CEA> 28A9 Dotam-VH-AST (D1AE3671) 94Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val
Leu Val465 470 475 480Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser 485 490 495Leu Ser Thr Tyr Ser Met Ser Trp Ile
Arg Gln Pro Pro Gly Lys Ala 500 505 510Leu Glu Trp Leu Gly Phe Ile
Gly Ser Arg Gly Asp Thr Tyr Tyr Ala 515 520 525Ser Trp Ala Lys Gly
Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser 530 535 540Gln Val Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr545 550 555
560Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro
565 570 575Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
Ala Ser 580 585 590Thr95449PRTArtificial SequenceP1AF711 HCknob
<CEA> 28A9 without linker and DOTAM 95Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Tyr Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Leu Pro Ala Phe Gly Ala Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Pro Pro Leu Pro Gly Ala Gly Leu Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly96214PRTArtificial SequenceP1AF710 and P1AF711 light chain
(D1AA2299) 96Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Asn Thr Gln Tyr Pro Met 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21097450PRTArtificial SequenceP1AF0712 HCknob <CEA> CH1A1A
(D1AC4023) 97Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala
Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala
Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45098579PRTArtificial SequenceP1AF0712 HChole <CEA> CH1A1A
DOTA-VL (D1AE4684) 98Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly
Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr
Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp
Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser
Gly Gly Gly Gly Ser Gln Ala Val Val Ile Gln Glu Ser Ala Leu465 470
475 480Thr Thr Pro Pro Gly Glu Thr Val Thr Leu Thr Cys Gly Ser Ser
Thr 485 490 495Gly Ala Val Thr Ala Ser Asn Tyr Ala Asn Trp Val Gln
Glu Lys Pro 500 505 510Asp His Leu Phe Thr Gly Leu Ile Gly Gly His
Asn Asn Arg Pro Pro 515 520 525Gly Val Pro Ala Arg Phe Ser Gly Ser
Leu Ile Gly Asp Lys Ala Ala 530 535 540Leu Thr Ile Ala Gly Thr Gln
Thr Glu Asp Glu Ala Ile Tyr Phe Cys545 550 555 560Ala Leu Trp Tyr
Ser Asp His Trp Val Ile Gly Gly Gly Thr Lys Leu 565 570 575Thr Val
Leu99450PRTArtificial SequenceP1AF0712 HChole <CEA> without
linker or DOTA 99Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu
Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe
Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
450100450PRTArtificial SequenceP1AF0713 HCHole <CEA> CH1A1A
(D1AC4022) 100Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala
Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala
Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
450101592PRTArtificial SequenceP1AF0713 HCknob <CEA> CH1A1A
DOTA-VH-AST (D1AE3670) 101Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr
Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp
Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser His Val Lys Leu Gln Glu Ser Gly Pro Gly465 470 475
480Leu Val Gln Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly
485 490 495Phe Ser Leu Thr Asp Tyr Gly Val His Trp Val Arg Gln Ser
Pro Gly 500 505 510Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly
Gly Gly Thr Ala 515 520 525Tyr Asn Thr Ala Leu Ile Ser Arg Leu Asn
Ile Tyr Arg Asp Asn Ser 530 535 540Lys Asn Gln Val Phe Leu Glu Met
Asn Ser Leu Gln Ala Glu Asp Thr545 550 555 560Ala Met Tyr Tyr Cys
Ala Arg Arg Gly Ser Tyr Pro Tyr Asn Tyr Phe 565 570 575Asp Ala Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 580 585
590102450PRTArtificial SequenceP1AF0713 HCknob <CEA> CH1A1A
without linker and DOTA 102Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr
Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp
Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
450103215PRTArtificial SequenceP1AF0712 and P1AF0713 light chain
(D1AA3384) 103Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala
Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys His Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 215104447PRTArtificial SequenceP1AF8284 and P1AF8285 HCknob
<GPRC5D> (D1AF6517) 104Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr
20 25 30Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr Met 100 105 110Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Cys Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Trp Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445105359PRTArtificial SequenceP1AF8284 HChole Dotam-VL
(D1AG3592) 105Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135
140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly225 230 235 240Gly
Ser Gly Gly Gly Gly Ser Ser Ile Gln Met Thr Gln Ser Pro Ser 245 250
255Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser
260 265 270Ser His Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln
Gln Lys 275 280 285Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala
Ser Lys Leu Ala 290 295 300Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe305 310 315 320Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 325 330 335Cys Leu Gly Gly Tyr
Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly 340 345 350Gly Thr Lys
Val Glu Ile Lys 355106369PRTArtificial SequenceP1AF8285 Hhole
Dotam-VHA (D1AG3591) 106Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120
125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly225 230 235
240Gly Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val
245 250 255Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val
Ser Gly 260 265 270Phe Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg
Gln Pro Pro Gly 275 280 285Lys Ala Leu Glu Trp Leu Gly Phe Ile Gly
Ser Arg Gly Asp Thr Tyr 290 295 300Tyr Ala Ser Trp Ala Lys Gly Arg
Leu Thr Ile Ser Lys Asp Thr Ser305 310 315 320Lys Ser Gln Val Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr 325 330 335Ala Thr Tyr
Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala 340 345 350Tyr
Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 355 360
365Ala107218PRTArtificial SequenceP1AF8284 and P1AF8285 light chain
(D1AF6469) 107Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ile Ser 20 25 30Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys
Pro Gly Gln Gln Pro 35 40 45Lys Leu Leu Ile Tyr His Ala Ser Ile Leu
Ala Ser Gly Ile Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser65 70 75 80Arg Leu Glu Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Thr Arg 85 90 95Glu Ser Pro Leu Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 210 215108447PRTArtificial SequenceP1AF8286 and
P1AF8287 HCknob <FAP> 4B9 (D1AF6515) 108Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Trp Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445109359PRTArtificial SequenceP1AF8286 HChole Dotam-VL (D1AG3592)
109Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly225 230 235 240Gly Ser Gly Gly Gly
Gly Ser Ser Ile Gln Met Thr Gln Ser Pro Ser 245 250 255Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser 260 265 270Ser
His Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys 275 280
285Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala
290 295 300Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe305 310 315 320Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr 325 330 335Cys Leu Gly Gly Tyr Asp Asp Glu Ser
Asp Thr Tyr Gly Phe Gly Gly 340 345 350Gly Thr Lys Val Glu Ile Lys
355110369PRTArtificial SequenceP1AF8287 HChole Dotam-VHA (D1AG3591)
110Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly225
230 235 240Gly Ser Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly
Pro Val 245 250 255Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys
Thr Val Ser Gly 260 265 270Phe Ser Leu Ser Thr Tyr Ser Met Ser Trp
Ile Arg Gln Pro Pro Gly 275 280 285Lys Ala Leu Glu Trp Leu Gly Phe
Ile Gly Ser Arg Gly Asp Thr Tyr 290 295 300Tyr Ala Ser Trp Ala Lys
Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser305 310 315 320Lys Ser Gln
Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr 325 330 335Ala
Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala 340 345
350Tyr Pro Pro His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
355 360 365Ala111215PRTArtificial SequenceP1AF8286 and P1AF8287
light chain (D1AB9974) 111Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Thr Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Asn Val Gly Ser Arg Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90 95Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly
Glu Cys 210 215112451PRTArtificial SequenceP1AF7782 and P1AF7784
HCknob <CEA> CH1A1A (D1AD3419) 112Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn
Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg
Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Lys 450113359PRTArtificial SequenceP1AF7782 HChole Dotam-VL
(D1AG2237) 113Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser
Val Tyr Ser Asp 20 25 30Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu65 70 75 80Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp 85 90 95Glu Ser Asp Thr Tyr Gly
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Ser
Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135
140Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys145 150 155 160Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 165 170 175Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp 180 185 190Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu225 230 235 240Gly
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250
255Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp 275 280 285Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 290 295 300Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Val Ser305 310 315 320Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350Leu Ser Leu
Ser Pro Gly Lys 355114369PRTArtificial SequenceP1AF7784 HChole
Dotam-VH (D1AG2236) 114Gly Val Thr Leu Lys Glu Ser Gly Pro Val Leu
Val Lys Pro Thr Glu1 5 10 15Thr Leu Thr Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Ser Thr Tyr 20 25 30Ser Met Ser Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu Trp Leu 35 40 45Gly Phe Ile Gly Ser Arg Gly Asp
Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60Gly Arg Leu Thr Ile Ser Lys
Asp Thr Ser Lys Ser Gln Val Val Leu65 70 75 80Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Arg Glu Arg Asp
Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp 100 105 110Gly Arg
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120
125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Asp Lys
130 135 140Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly Pro145 150 155 160Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 165 170 175Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 180 185 190Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 195 200 205Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 210 215 220Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu225 230 235
240Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
245 250 255Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Cys Thr 260 265 270Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Ser 275 280 285Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 290 295 300Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu305 310 315 320Asp Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 325 330 335Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 340 345 350Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 355 360
365Lys115215PRTArtificial SequenceP1AF7782 and P1AF7784 light chain
(D1AD3421) 115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala
Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys His Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 21511610PRTArtificial Sequenceheavy chain CDR1
<Pb-Dotam> PRIT-0213 116Gly Phe Ser Leu Ser Thr Tyr Ser Met
Ser1 5 1011716PRTArtificial Sequenceheavy chain CDR2
<Pb-Dotam> PRIT-0213 117Phe Ile Gly Ser Arg Gly Asp Thr Tyr
Tyr Ala Ser Trp Ala Lys Gly1 5 10 1511814PRTArtificial
Sequenceheavy chain CDR3 <Pb-Dotam> PRIT-0213 118Glu Arg Asp
Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu1 5 1011913PRTArtificial
Sequencelight chain CDR1 <Pb-Dotam> PRIT-0213 119Gln Ser Ser
His Ser Val Tyr Ser Asp Asn Asp Leu Ala1 5 101207PRTArtificial
Sequencelight chain CDR2 <Pb-Dotam> PRIT-0213 120Gln Ala Ser
Lys Leu Ala Ser1 512112PRTArtificial Sequencelight chain CDR3
<Pb-Dotam> PRIT-0213 121Leu Gly Gly Tyr Asp Asp Glu Ser Asp
Thr Tyr Gly1 5 10122121PRTArtificial Sequenceheavy chain variable
domain 1 of <Pb-Dotam> PRIT-0213 122Val Thr Leu Lys Glu Ser
Gly Pro Val Leu Val Lys Pro Thr Glu Thr1 5 10 15Leu Thr Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 35 40 45Phe Ile Gly
Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Leu
Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr65 70 75
80Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
85 90 95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120123111PRTArtificial Sequencelight chain variable domain
<Pb-Dotam> PRIT-0213 123Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly Asp1 5 10 15Arg Val Thr Ile Thr Cys Gln Ser
Ser His Ser Val Tyr Ser Asp Asn 20 25 30Asp Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gln Ala Ser Lys
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu 85 90 95Ser Asp
Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110124121PRTArtificial Sequenceheavy chain variable domain
<Pb-Dotam> PRIT-0214 124Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu Thr1 5 10 15Leu Ser Leu Thr Cys Ala Val Tyr
Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45Phe Ile Gly Ser Arg Gly
Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Val Thr Ile Ser
Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys65 70 75 80Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95Glu Arg
Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly 100 105
110Arg Gly Thr Leu Val Thr Val Ser Ser 115 120125111PRTArtificial
Sequencelight chain variable domain <Pb-Dotam> PRIT-0214
125Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp1
5 10 15Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp
Asn 20 25 30Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gly Gly Tyr Asp Asp Glu 85 90 95Ser Asp Thr Tyr Gly Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 110126591PRTArtificial
Sequenceheavy chain 1 of bispecific, trivalent <CEA/Pb-Dotam>
PRIT-0214 VH_84.66 126Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5
10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val
Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp
Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Trp Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly Gly Gly Ser Val Gln
Leu Gln Gln Trp Gly Ala Gly Leu465 470 475 480Leu Lys Pro Ser Glu
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe 485 490 495Ser Leu Ser
Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys 500 505 510Gly
Leu Glu Trp Ile Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr 515 520
525Ala Ser Trp Ala Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys
530 535 540Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala545 550 555 560Val Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr
Gly Gly Gly Ala Tyr 565 570 575Pro Pro His Leu Trp Gly Arg Gly Thr
Leu Val Thr Val Ser Ser 580 585 590127581PRTArtificial
Sequenceheavy chain 2 of bispecific, trivalent<CEA/Pb-Dotam>
PRIT-0214 VL_84.66 127Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly
Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly
Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu465 470 475
480Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His
485 490 495Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp Tyr Gln Gln Lys
Pro Gly 500 505 510Lys Ala Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys
Leu Ala Ser Gly 515 520 525Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu 530 535 540Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Leu545 550 555 560Gly Gly Tyr Asp Asp
Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr 565 570 575Lys Val Glu
Ile Lys 580128218PRTArtificial Sequencelight chain <CEA>
84.66 128Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val
Asp Ile Phe 20 25 30Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215129591PRTArtificial Sequenceheavy chain 1 of bispecific,
trivalent <CEA/Pb-Dotam> PRIT-0213 VH_84.66 --> knob
129Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val
Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp
Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Cys Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Trp Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 450 455 460Ser Gly Gly Gly Gly Ser Val Thr
Leu Lys Glu Ser Gly Pro Val Leu465 470 475 480Val Lys Pro Thr Glu
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe 485 490 495Ser Leu Ser
Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys 500 505 510Ala
Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr 515 520
525Ala Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
530 535 540Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala545 550 555 560Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr
Gly Gly Gly Ala Tyr 565 570 575Pro Pro His Leu Trp Gly Arg Gly Thr
Leu Val Thr Val Ser Ser 580 585 590130581PRTArtificial
Sequenceheavy chain 2 of bispecific, trivalent <CEA/Pb-Dotam>
PRIT-0213 VL_84.66 -->hole 130Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro
Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455
460Ser Gly Gly Gly Gly Ser Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu465 470 475 480Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Gln Ser Ser His 485 490 495Ser Val Tyr Ser Asp Asn Asp Leu Ala Trp
Tyr Gln Gln Lys Pro Gly 500 505 510Lys Ala Pro Lys Leu Leu Ile Tyr
Gln Ala Ser Lys Leu Ala Ser Gly 515 520 525Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 530 535 540Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu545 550 555 560Gly
Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr 565 570
575Lys Val Glu Ile Lys 580131581PRTArtificial Sequenceheavy chain 1
of bispecific, <CEA/Pb-Dotam> Rabbit Dotam _84.66 131Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala
Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly 450 455 460Ser Gly Gly Gly Gly Ser Ala Val Leu Thr
Gln Thr Pro Ser Pro Val465 470 475 480Ser Pro Ala Val Gly Gly Thr
Val Thr Ile Ser Cys Gln Ser Ser His 485 490 495Ser Val Tyr Ser Asp
Asn Asp Leu Ala Trp Tyr Gln Gln Lys Leu Gly 500 505 510Gln Pro Pro
Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly 515 520 525Val
Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 530 535
540Thr Ile Ser Gly Val Gln Ser Asp Asp Ala Ala Thr Tyr Tyr Cys
Leu545 550 555 560Gly Gly Tyr Asp Asp Glu Ser Asp Thr Tyr Gly Phe
Gly Gly Gly Thr 565 570 575Glu Val Val Val Lys
580132121PRTArtificial SequenceHC5 132Val Thr Leu Lys Glu Ser Gly
Pro Val Leu Val Lys Pro Thr Glu Thr1 5 10 15Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 35 40 45Phe Ile Gly Ser
Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr65 70 75 80Met
Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 85 90
95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly
100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120133112PRTArtificial SequenceLC1 133Ser Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Gln Ser Ser His Ser Val Tyr Ser Asp 20 25 30Asn Asp Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gln
Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu65 70 75 80Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp 85 90
95Glu Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110134111PRTArtificial SequenceLC3 134Ala Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp1 5 10 15Arg Val Thr Ile
Thr Cys Gln Ser Ser His Ser Val Tyr Ser Asp Asn 20 25 30Asp Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45Ile Tyr
Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75
80Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Asp Glu
85 90 95Ser Asp Thr Tyr Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110135121PRTArtificial SequenceHC7 135Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Phe Ile
Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg
Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln65 70 75
80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120136121PRTArtificial SequenceHC10 136Val Gln Leu Gln Gln Trp Gly
Ala Gly Leu Leu Lys Pro Ser Glu Thr1 5 10 15Leu Ser Leu Thr Cys Ala
Val Tyr Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45Phe Ile Gly Ser
Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Val Thr
Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys65 70 75 80Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90
95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu Trp Gly
100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120137121PRTArtificial SequenceT84.66 VH 1 137Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120138111PRTArtificial SequenceT84.66 VL 138Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30Gly Val Gly Phe
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu
Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110139121PRTArtificial SequenceCH1A1A VH 139Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55
60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr
Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
120140108PRTArtificial SequenceCH1A1A VL 140Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu
85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10514137DNAArtificial SequencerbHC.up primer sequence 141aagcttgcca
ccatggagac tgggctgcgc tggcttc 3714221DNAArtificial
Sequencehttps//protect-eu.mimecast.com/s/
jhrhC2RNxFYMPVS2Ygk4?domain=rbhcf.do primer sequence 142ccattggtga
gggtgcccga g 2114334DNAArtificial SequencerbLC.up Primer sequence
143aagcttgcca ccatggacay gagggccccc actc 3414426DNAArtificial
Sequencehttps//protect-eu.mimecast.com/s/
2MppC3lXQckn6XCQnhR8?domain=rblc.do Primer sequence 144cagagtrctg
ctgaggttgt aggtac 26145224PRTArtificial SequenceP1AA1227_HC 145Val
Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr1 5 10
15Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser
20 25 30Met Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
Gly 35 40 45Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala
Lys Gly 50 55 60Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val
Val Leu Thr65 70 75 80Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr Cys Ala Arg 85 90 95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr
Pro Pro His Leu Trp Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220146219PRTArtificial SequenceP1AA1227_LC
146Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ser Ser His Ser Val Tyr Ser
Asp 20 25 30Asn Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu 35 40 45Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu65 70 75 80Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gly Gly Tyr Asp Asp 85 90 95Glu Ser Asp Thr Tyr Gly Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215147594PRTArtificial SequenceD1AE3669 (HCknob <CEA>
CH1A1A Dotam-VH-AST) 147Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly
Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr
Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp
Phe Ala Tyr Tyr Val Glu
Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly 450 455 460Ser Gly Gly Gly Gly Ser Val Thr Leu Lys
Glu Ser Gly Pro Val Leu465 470 475 480Val Lys Pro Thr Glu Thr Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe 485 490 495Ser Leu Ser Thr Tyr
Ser Met Ser Trp Ile Arg Gln Pro Pro Gly Lys 500 505 510Ala Leu Glu
Trp Leu Gly Phe Ile Gly Ser Arg Gly Asp Thr Tyr Tyr 515 520 525Ala
Ser Trp Ala Lys Gly Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 530 535
540Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr
Ala545 550 555 560Thr Tyr Tyr Cys Ala Arg Glu Arg Asp Pro Tyr Gly
Gly Gly Ala Tyr 565 570 575Pro Pro His Leu Trp Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Ala 580 585 590Ser Thr14810PRTArtificial
SequenceCDR H1 148Gly Phe Ser Leu Thr Asp Tyr Gly Val His1 5
1014910PRTArtificial Sequencehuman IgG1 CH1 domain 149Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro1 5 10150122PRTArtificial Sequenceheavy
chain variable domain with c terminus extension 150Val Thr Leu Lys
Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr1 5 10 15Leu Thr Leu
Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr Ser 20 25 30Met Ser
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 35 40 45Phe
Ile Gly Ser Arg Gly Asp Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55
60Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr65
70 75 80Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
Arg 85 90 95Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr Pro Pro His Leu
Trp Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser Ala 115
12015110PRTArtificial SequenceCDR H1 151Gly Phe Thr Phe Thr Asp Tyr
Tyr Met Asn1 5 1015220PRTArtificial Sequenceflexible linker 152Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10
15Gly Ser Gly Gly 20153592PRTArtificial Sequencea variant with
c-terminal alanine extension 153Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys
Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr
Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460Ser
Gly Gly Gly Gly Ser Val Thr Leu Lys Glu Ser Gly Pro Val Leu465 470
475 480Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly
Phe 485 490 495Ser Leu Ser Thr Tyr Ser Met Ser Trp Ile Arg Gln Pro
Pro Gly Lys 500 505 510Ala Leu Glu Trp Leu Gly Phe Ile Gly Ser Arg
Gly Asp Thr Tyr Tyr 515 520 525Ala Ser Trp Ala Lys Gly Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys 530 535 540Ser Gln Val Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala545 550 555 560Thr Tyr Tyr Cys
Ala Arg Glu Arg Asp Pro Tyr Gly Gly Gly Ala Tyr 565 570 575Pro Pro
His Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala 580 585
59015488PRTArtificial SequenceA5B7 epitope 154Pro Lys Pro Phe Ile
Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu1 5 10 15Asp Ala Val Ala
Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr 20 25 30Leu Trp Trp
Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 35 40 45Leu Ser
Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn 50 55 60Asp
Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser Val Asp65 70 75
80His Ser Asp Pro Val Ile Leu Asn 8515588PRTArtificial
SequenceMFE23 epitope 155Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser
Lys Pro Val Glu Asp Lys1 5 10 15Asp Ala Val Ala Phe Thr Cys Glu Pro
Glu Thr Gln Asp Ala Thr Tyr 20 25 30Leu Trp Trp Val Asn Asn Gln Ser
Leu Pro Val Ser Pro Arg Leu Gln 35 40 45Leu Ser Asn Gly Asn Arg Thr
Leu Thr Leu Phe Asn Val Thr Arg Asn 50 55 60Asp Thr Ala Ser Tyr Lys
Cys Glu Thr Gln Asn Pro Val Ser Ala Arg65 70 75 80Arg Ser Asp Ser
Val Ile Leu Asn 851565PRTArtificial Sequenceheavy chain CDR1
<CEA> MFE23 156Asp Ser Tyr Met His1 515717PRTArtificial
Sequenceheavy chain CDR2 <CEA> MFE23 157Trp Ile Asp Pro Glu
Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln1 5 10
15Gly15817PRTArtificial Sequenceheavy chain CDR2 <CEA>
MFE23-H26 158Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln
Lys Phe Gln1 5 10 15Gly15911PRTArtificial Sequenceheavy chain CDR3
<CEA> MFE23 159Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr1 5
1016010PRTArtificial Sequencelight chain CDR1 <CEA> MFE23
160Ser Ala Ser Ser Ser Val Ser Tyr Met His1 5 1016110PRTArtificial
Sequencelight chain CDR1 <CEA> MFE23-L24, L25 161Arg Ala Ser
Ser Ser Val Ser Tyr Met His1 5 1016220PRTArtificial Sequencelight
chain CDR1 <CEA> MFE23-L26 162Arg Ala Ser Gln Ser Ile Ser Ser
Tyr Met Arg Ala Ser Gln Ser Ile1 5 10 15Ser Ser Tyr Met
201637PRTArtificial Sequencelight chain CDR2 <CEA> MFE23
163Ser Thr Ser Asn Leu Ala Ser1 51647PRTArtificial SequenceLight
chain CDR2 <CEA> MFE23-L26 164Tyr Thr Ser Asn Leu Ala Ser1
51657PRTArtificial SequenceLight chain CDR2 <CEA> MFE23-L29
165Ser Thr Ser Ser Leu Gln Ser1 51669PRTArtificial Sequencelight
chain CDR3 <CEA> MFE23 166Gln Gln Arg Ser Ser Tyr Pro Leu
Thr1 5167120PRTArtificial SequenceHeavy chain variable domain
<CEA> MFE23 167Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Ser Gly Thr1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp Leu Arg Gln Gly Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly
Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Phe Thr
Thr Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Asn Glu Gly Thr
Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 120168106PRTArtificial SequenceLight
chain variable domain <CEA> MFE23 168Glu Asn Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Ile
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Phe Gln
Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75
80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105169120PRTArtificial SequenceMFE-H24 169Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120170120PRTArtificial SequenceMFE-H25 170Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120171120PRTArtificial SequenceMFE-H26 171Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120172120PRTArtificial SequenceMFE-H27 172Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120173120PRTArtificial SequenceMFE-H28 173Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5
10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp
Ser 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala
Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr
Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120174120PRTArtificial SequenceMFE-H29 174Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120175106PRTArtificial SequenceMFE-L24 175Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105176106PRTArtificial SequenceMFE-L25 176Glu Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105177107PRTArtificial SequenceMFE-L26 177Glu Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Met His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105178106PRTArtificial SequenceMFE-L27 178Glu Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105179106PRTArtificial SequenceMFE-L28 179Glu Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met 20 25 30His Trp Leu Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105180106PRTArtificial SequenceMFE-L29 180Glu Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met 20 25 30His Trp Leu Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
* * * * *
References